Treatment of retinal degeneration using gene therapy

ABSTRACT

The present invention relates to an improved method of providing photoreceptor function to a cell, for example for use in the treatment of retinal degeneration. The present invention also relates to compositions and kits, in particular for use in such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/121,283filed Aug. 24, 2016, which is the National Phase of InternationalApplication No. PCT/GB2015/050516 filed Feb. 24, 2015, which designatedthe U.S. and that International Application was published under PCTArticle 21(2) in English. All applications include a claim of priorityunder 35 U.S.C. § 119(a) and § 365(b) to British patent application No.GB 1403260.1 filed Feb. 25, 2014, the entirety of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to an improved method of providingphotoreceptor function to a cell, for example for use in the treatmentof retinal degeneration. The present invention also relates tocompositions and kits, in particular for use in such methods.

BACKGROUND

The retina of the vertebrate eye serves the same function as a film in acamera, receiving a visual image created by light passing through thelens and cornea of the eye. The image received is translated intochemical and electrical signals which are transmitted to the brain viathe optic nerve.

The retina is a complex structure, comprising ten distinct layers ofdifferent cell types. Of these layers, it is the photoreceptor layerwhich is responsible for translating the incoming light into a chemicaland/or electrical signal which can be read by the brain and interpretedinto an image. The photoreceptor layer comprises photosensitive cells oftwo types, known as rod cells and cone cells. These cell types are bothresponsible for reacting to incoming light and producing an electricalsignal, but differ in in their positioning within the retina and thetype of light which they react to. Specifically, rod cells functionmostly in dim light and are found predominantly in the peripheralretina. Cone cells are more reactive to bright light (i.e. daytimevision) are responsible for colour vision and are found at highestdensity in the central retina. The retina also contains a third lessnumerous type of photoreceptor cell—photosensitive ganglion cells—whichare responsible for measuring background light, but not imageprocessing.

The rod and cone cells of the photoreceptor layer of the retina are ableto react to light and convert it to an electrical signal due to thepresence of photosensitive pigments (referred to as photopigments)therein, which undergo a chemical change when the cell is exposed tolight. These photopigments are G-protein-coupled receptors. Thephotopigments comprise a protein moiety which is coupled to achromophoric cofactor known as retinal. Exposure to light causes anisomerisation of the retinal cofactor from a cis-retinal to atrans-retinal, which in turn causes a conformational change in the opsinprotein, known as photobleaching. This is the first step in a signalingcascade which results in a signal being transmitted along the opticnerve. In order to retain photosensitivity, opsins thus need acontinuous supply of cis retinal. Neither rod nor cone photoreceptorcells are able to produce cis-retinal themselves. The major source ofcis-retinal in the retina is the RPE (retinal pigment epithelium) whichtakes all trans-retinal from bleached opsin and produces cis-retinal. Inthe intact retina, rod and cone cells abut the RPE allowing them accessto this regenerated chromophore.

In humans, several closely related photopigments exist, known as theopsin family. In humans, these comprise 3 cone opsins which aresensitive to different wavelengths (the origin of colour vision),rhodopsin found in rod cells, and melanopsin found in ganglion cellphotoreceptors. The cone opsins include LWS opsin for yellowish-green,MWS opsin for green, and SWS opsin for bluish-violet. These opsins showhigh sequence identity.

Conditions such as retinal dystrophies cause blindness due todestruction of the photoreceptors in the outer retina (i.e. the rods andcones). These conditions may be a result of direct damage to thephotoreceptors, or photoreceptors being indirectly destroyed as a resultof pathology in the retinal pigment epithelium and/or choroid. Severevisual impairment is common in advanced stages of the degeneration.These conditions are currently incurable. Retinal dystrophies can bedivided into rod-cone dystrophies (also called retinitis pigmentosa),cone-rod dystrophies and macular dystrophies. In rod-cone dystrophiesthe rod photoreceptors degenerate resulting in a loss of peripheralvision and night vision, and frequently this is followed by conedestruction leading to a loss of central and colour vision. Converselyin the cone-rod dystrophies there is initially a loss of conephotoreceptors leading to a loss of detailed and colour vision and thisis then followed by rod degeneration resulting in a loss of peripheralvision and night blindness. Both forms can result in blindness withextensive or complete loss of visual field. Another type of retinaldystrophy called macular dystrophy results in a loss of central vision,but peripheral vision is preserved.

However, despite the loss of outer retinal photoreceptors, inner retinalneurons, including bipolar cells and retinal ganglion cells, can surviveand retain their ability to send visual information to the brain. Theseneurons therefore, provide a promising niche for emerging optogenetictherapies that aim to convert them into directly visual photoreceptorsand recreate the photosensitivity that has been lost with thedegeneration. Several therapeutic strategies have shown promisingresults in attempts to replace or revive these inner retinal neurons andrestore vision. Transplantation of photoreceptor cells, or theirprogenitor lines, is a major approach under pre-clinical study and hasbeen shown to restore vision to blind mice at late stage of degenerationafter complete loss of photoreceptors. In attempts to revive innerretinal neurons, implantable electronic prostheses have triggeredretinal ganglion cell (RGC) firing through external cameras and haveprovided crude spatial discrimination for at least some patients(Zrenner E, et al. 2011, Proc Biol Sci 278(1711):1489-1497; Humayun M S,et al. 2012, Ophthalmology 119, 779-788). Another strategy usesmicrobial opsins as photoswitches of neuronal activity and they havebeen used to elicit light-evoked activity in degenerate retinas. To thisextent it has been shown that intravitreal injection of an AAV-2 vectorcarrying the channelrhodopsin-2 gene (ChR2) in the rd1 mouse leads tolight activated depolarization or ‘ON’ responses in RGCs and visuallyevoked potentials in the cortex. This study led by Bi et al. (Bi A, etal 2006, Neuron. 2006; 6; 50(1):23-33.)) provided the firstproof-of-principle that retinal function can be restored usingoptogenetics. (Lagali P S, et al 2008, Nat Neurosci. 2008 June;11(6):667-75; Cronin T, et al 2014, EMBO Mol Med. 2014 Aug. 4;6(9):1175-90; Mace E et al 2014, Mol Ther. 2015 January; 23(1):7-16) aswell as cone photoreceptors (Busskamp V, et al 2010, Science. 2010 Jul.23; 329(5990):413-7.) have been successfully converted to artificiallight-sensors leading to partial rescue of visual function in blindmice. In addition, recently developed synthetic photoswitches have shownpromising results in rescuing vision in blind mice. The ‘one-component’azobenzene-based photoswitches use small molecules, AAQ or DENAQ(Polosukhina A, et al 2012, Neuron. 2012 Jul. 26; 75(2):271-82;Tochitsky I, et al 2014, Neuron. 2014 Feb. 19; 81(4):800-13.) thatdirectly photosensitise native ion channels of neurons. The‘two-component’ photoswitches, LiGluR/MAG, (Caporale N, et al 2011, MolTher. 2011 July; 19(7):1212-9; Gaub B M, et al 2014, Proc Natl Acad SciUSA. 2014 Dec. 23; 111(51):E5574-83) first genetically expresssynthetically engineered light-gated ionotropic glutamate receptor(LiGluR) in retinas and then require addition of a photoswitch molecule(MAG) for activation. Both systems have shown to impart lightsensitivity to blind mouse and canine (Gaub B M, et al 2014, Proc NatlAcad Sci USA. 2014 Dec. 23; 111(51):E5574-83) retinas and restore basicvisual functions in rodents.

However improvements are still necessary in the treatment of theseconditions.

The present invention aims to overcome or ameliorate the problemsassociated with the treatment of retinal degeneration.

BRIEF SUMMARY OF THE INVENTION

Thus, in a first aspect of the present invention, there is provided amethod of providing photoreceptor function to a cell, the methodcomprising introducing into the vitreal cavity of an eye i) a nucleicacid sequence encoding a photosensitive protein; and ii) anextracellular matrix degradation enzyme.

In a second aspect of the present invention, there is provided acomposition comprising i) a nucleic acid sequence encoding aphotosensitive protein and ii) an extracellular matrix degradationenzyme. Preferably, the composition is therapeutic.

In a third aspect of the present invention, there is provided i) anucleic acid sequence encoding a photosensitive protein; and ii) anextracellular matrix degradation enzyme, for use in a method ofproviding photoreceptor function to a cell.

In a fourth aspect of the invention, there is provided a method ofproviding photoreceptor function to a cell, the method comprisingintroducing into an eye a nucleic acid vector comprising a nucleic acidencoding a human photoreceptor protein. In this aspect, preferably thevector is introduced without administration of an extracellular matrixdegradation enzyme (for example, it is introduced withoutco-administration of an enzyme, wherein co-administration includesseparate, sequential or combined administration during the sametherapy). The method may further comprise expressing the vector in innerretinal cells, wherein expression of the human photoreceptor proteinrenders an inner retinal cell photoreceptive.

In a fifth aspect of the invention, there is provided a nucleic acidvector comprising a nucleic acid encoding a human photoreceptor protein.

DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter withreference to the accompanying drawings, in which:

FIG. 1 shows intraocular injections and gene delivery via AAV;

FIG. 2 shows restoration of pupillary light reflex following rhodopsintreatment;

FIG. 3A-3E shows data from in-vivo electrophysiology recordings; FIG.3F) Heat map of representative in-vivo dLGN light responses from Rd1mice (n=5) where one eye was treated with rod opsin, CAG-hRho (FIG. 3B)and the other with GFP, CAG-GFP (FIG. 3C) depicting the diversity ofrestored responses: sustained, transient, ON and OFF. Responses wereordered according to the amplitude of sustained response during theinitial 1 second of a 2 second light stimulus presentation (15.4 logphotons/cm2/s); Figure G-J) Representative peri-stimulus time histograms(PSTHs) showing average response to multiple presentations of full fieldflashes (15.4 log photons/cm2/s) for ON-sustained (FIG. 3G),ON-transient (FIG. 3H), OFF (FIG. 3I) and ON-OFF (FIG. 3J).Corresponding trial bin counts (TBCs) are shown on top of each PSTH.

FIG. 4A-4C shows data from in-vitro MEA recordings;

FIG. 5 shows the observed responses are clearly distinct from nativelight responses normally present in rd1 mouse retina following lightstimulation.

FIG. 6 shows dLGN responses in rd1 mouse driven by rod opsin treatedeyes respond over a range of light intensities and in light adaptedconditions FIG. 6A) Sensitivity response profile (PSTH and TBC) of fiverepresentative dLGN units from Rd1-CAG-hRho mice presented with fullfield flashes at different light intensities (ND2=13.4, ND1=14.4 andND0=15.4 log photons/cm2/s); FIG. 6B) Contrast sensitivity responseprofile (PSTH and TBC) of four representative dLGN units fromRd1-CAG-hRho mice recorded under light adapted conditions (Michelsoncontrast 96% @ ND0=15.4 log photons/cm2/s).

FIG. 7 shows targeted expression of rod opsin to ON bipolar cellsrestores visual responses in blind rd1 retinas: FIG. 7A) AAV2 vector DNAconstruct driving targeted expression of human rod opsin under Grm6promoter; FIGS. 7B and 7C) Exemplar fluorescent microscope images of asection through the mouse retina after intravitreal delivery of viralvector in A in conjunction with glycosidic enzymes that break down theextracellular matrix (FIG. 7B). High magnification fluorescent imagesdepicting membranous localization of rod opsin in cell somas of INLcells (FIG. 7C). Retinas were treated with α-hRho antibody (red) andnuclei were stained with DAPI (blue). Calibration bar=50 μm.GCL=ganglion cell layer, IPL=inner plexiform layer, INL=inner nuclearlayer.

FIG. 8A-8B shows representative peri-stimulus time histograms (PSTHs)showing average responses to multiple presentations of 5-second fullfield flashes @ND0=15.4 log photons/cm2/s for ON (FIG. 8A) and OFF (FIG.8B) responses.

FIG. 9 shows representative peri-stimulus time histograms (PSTHs)showing average responses to multiple presentations of 2-second fullfield flashes @ lower light levels (ND2-13.4 log photons/cm2/s).

FIG. 10 shows representative peri-stimulus time histograms (PSTHs)showing average responses to multiple presentations of 5-second fullfield flashes under light adapted conditions—Michelson contrast 96%.

FIG. 11A-11D shows ectopic expression of rod opsin restores visualbehaviour in treated blind rd1 mice: FIG. 11A) Irradiance-responsecurves for maximum pupillary constriction during 10 s of white light ata range of light intensities (FIG. 11A). Rod opsin treated rd1 eyes(untargeted CAG expression, red) show a marked improvement in visualsensitivity compared to GFP injected rd1 eyes (green). With targetedexpression (Grm6, blue) the pupillary light reflex remained largelyimpaired. Data for wild-type mice injected with PBS/enzyme mixture areshown for comparison (black). Data are normalised to pupil sizeimmediately preceding the onset of light. Values are mean±SEM, with nindicating the number of animals examined; FIG. 11B) Representativeinfrared images of pupil area measured in the dark (baseline), at ND4(11.8 log photons/cm2/s) and at ND0 (15.8 log photons/cm2/s) for WT,Rd1-GFP, Rd1-CAG-hRho, Rd1-grm6-hRho mice; FIG. 11C, FIG. 11D) Meanmaximum pupillary constriction across the population of all four groupsof mice at ND4 (11.8 log photons/cm2/s; FIG. 11C) and ND0 (15.8 logphotons/cm2/s; FIG. 11D). Number of animals examined: WT n=6; Rd1-GFPn=16; Rd1-CAG-hRho n=10; Rd1-grm6-hRho n=6. Error bars are SEM.

FIG. 12A-12D shows open box activity plots from dark (shaded in dark) tolight (white area) for the four groups of mice: WT n=5 (FIG. 12A);Rd1-GFP n=6 (FIG. 12B); Rd1-CAG-hRho n=6 (FIG. 12C); Rd1-grm6-hRho n=5(FIG. 12D). Values in the plots are population means of distancetravelled in a preceding 30-second bin±SEM. Histograms on the right showmean distance travelled across the population for the transition periodfrom dark (30 s just before the light; dark bars) to light (30 s justafter the light; white bars). Error bars are SEM. *p<0.05, **p<0.005,paired Student t-test.

FIG. 13A) Open box activity plots from grey screen light to 4 Hz flickerlight for Rd1-grm6-hRho (n=5). Data are population mean of distancetravelled in a preceding 30-second bin±SEM. FIG. 13B) 4 Hz flickerresponse for Rd1-GFP (n=6), Rd1-CAG-hRho (n=6) and Rd1-grm6-hRho (n=5)mice. Paired histograms are shown for each group of mice depicting onlythe transition period from the entire activity plot (as shown forRd1-grm6-hRho) from grey light (30 s just before the 4 Hz flicker) to 4Hz flicker light (30 s just after the 4 Hz flicker). Data are populationmean of distance travelled±SEM. **p<0.005, paired Student t-test.

FIG. 14 shows Contrast sensitivity response for the Rd1-grm6-hRho mice(n=5) showing distance travelled before and after 4 Hz flicker atdifferent Michelson contrasts. For each contrast performance, only thetransition period from the activity plot is shown depicting the distancetravelled in the 30 s before (white bars) and in the 30 s after thepresentation of flicker (chequered bars). Data are population mean ofdistance travelled±SEM. *p<0.05, **p<0.005, paired Student t-test.

FIG. 15 shows flicker frequency response for the Rd1-grm6-hRho mice(n=5) showing distance travelled before and after presentation of fullfield flicker light at different frequencies. For each flicker response,only the transition period from the activity plot is shown comparing thedistance in the 30 s before (grey bars) to the distance in the 30 safter the presentation of flicker (patterned bars). Data are populationmean of distance travelled±SEM. *p<0.05, **p<0.005, paired Studentt-test.

FIG. 16A-16C shows targeted rod opsin expression restores responses tonaturalistic movie scenes: FIG. 16A, B) Raster plots for representativeresponsive dLGN units from Rd1-CAG-hRho (FIG. 16A) and Rd1-Grm6-hRho(FIG. 16B) mice exposed to multiple presentations of a 30 s naturalisticmovie (mice moving in an open arena in horizontal view); FIG. 16C) Openbox activity plots from grey screen light (shagged in grey) to a loomingowl movie (shaded in green) for Rd1-grm6-hRho (n=5). Data are populationmean of distance travelled in a preceding 30-second bin±SEM;

FIG. 17 shows naturalistic movie response for Rd1-GFP (n=6),Rd1-CAG-hRho (n=6) and Rd1-grm6-hRho (n=5) mice. Paired histograms areshown for each group of mice depicting only the transition period fromthe entire activity plot from grey light (30 s just before the moviepresentation; white bars) to owl movie (30 s just after the movie; blackbars). Data are population mean of distance travelled±SEM. **p<0.005,paired Student t-test.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of a human photoreceptorprotein to provide photoreceptor function to a cell, in order to restorephotosensitivity in degenerated or partially degenerated retinas. Suchnative photoreceptor function is lost with photoreceptor degeneration.The present invention is based upon the surprising finding thatexpression of a human photoreceptor protein in the inner retina canprovide photoreceptor function to the inner retinal neurone cells. Thetransgenic expression of a human photoreceptor protein in the innerretina has the advantage of minimising potential immunogenic adverseeffects, in contrast to the use of microbial opsins and electronicarrays respectively. In addition, outside photoreceptors, this GPCR haspotential to hijack cell's machinery and provide a completelyself-contained photoreceptive mechanism, capable of supporting lightdetection on its own and without any further interventions, unlikesynthetic photoswitches that require constant exogenous supply of aphotoswitch for activation. Moreover, rod opsin requires visible lightfor activation, and through the native GPCR amplification cascade it hasa potential to function under low light intensities in contrast tocurrent channel based or synthetic photoswitch systems, which are unableto amplify signals at the protein level.

Intravitreal injection as a method for gene therapy has particularadvantages in terms of being less technically challenging in access tothe retina, and reduced risk of complications during administration, inparticular where retinas have become thin through degradation.

The present invention is, in part, based upon the discovery thatco-administration of an extracellular matrix degradation enzyme in genetherapy leads to increased transduction of retinal cells, thus improvingthe outcome in terms of increased restoration of vision. The inventionrepresents an improvement over previous intra-vitreal gene therapymethods, by enabling increased transduction by reducing barriers tocontact of the foreign genetic material with the target cells.

The combined application of gene therapy and an extracellular matrixdegradation enzyme has led to surprising results in particular where thegene therapy comprises introduction of a nucleic acid sequence encodingrhodopsin to the vitreous of an eye. Rhodopsin requires the all-transretinal that is produced following visual transduction to be transportedto the retinal pigment epithelium (RPE) for it to be converted intoall-cis retinal which is then transported back to the rod cells forfurther visual response. This recycling has been thought to be dependentupon the intimate contact between rod cells and the RPE. Inner retinalcells, even in the presence of retinal dystrophies where the rods andcones degenerate, would not be physically associated with the RPE in thesame way. The present inventors have unexpectedly observed thatproviding rhodopsin to inner retinal cells by gene therapy produces avisual response, despite lack of contact between the functioning innerretinal cells and the RPE. Furthermore, it is thought that the innerretinal cells do not have the intracellular machinery required to workin conjunction with rhodopsin to produce an electrical signal that couldthen be transmitted via the ganglion cells to the brain.

The results are further surprising because rhodopsin works byhyperpolarising cells in response to light (the brains equivalent ofswitching the cells “off”), where it might be assumed that vision wouldrequire inner retinal cells to be depolarised (switched “on”) by light.

The present invention provides for the administration of anextracellular matrix degradation enzyme in combination with a nucleicacid sequence encoding a photosensitive protein to restorephotosensitive function to the retina. The retina and vitreous comprisea variety of extracellular matrix molecules, including proteoglycans(with different classes of glycosaminoglycan (GAG) chains) such asheparan sulphate proteoglycans (HSPGs), chondroitin sulphateproteoglycans (CSPGs), and dermatan sulphate proteoglycans (DSPGs);hyaluronan, collagens such as type IV collagen in the inner limitinglamina; laminins; nidogen 1 and 2, and a variety of other proteins andglycoproteins which are known to persons skilled in the art.

It is envisaged that the present invention may utilise any enzyme whichis capable of degrading an extracellular matrix protein or carbohydrate(such as glycosaminoglycan) present in the vitreous and/or in the retinaand/or internal limiting membrane, and/or retinal extracellular matrix.In particular, an enzyme for use in the present invention may be onewhich is capable of degrading an extracellular matrix protein orcarbohydrate which is provided in the retina, or an extracellular matrixprotein or carbohydrate which is provided in the path between thevitreous and the cells of the retina and which may therefore impact upontransduction of a nucleic acid sequence. Preferred are enzymes whichdegrade glycosaminoglycans (GAGs).

An extracellular matrix degradation protein may be selected from thegroup consisting of a collagenase, hyaluronan lyase, heparinase I,heparinase II, heparinase III, chondroitin ABC lyase, chondroitin AClyase, a metalloproteinase, an ADAMTS, a plasmin (serine proteaseplasmin or its truncated form microplasmin (Ocriplasmin)), neutrophilelastase and cathepsin G, neuraminidase, N-glycanase, 0-glycanase, andpronase. A particularly preferred enzyme may be selected from the groupconsisting of Hyaluronan lyase from Streptomyces hyalurolyticus (EC4.2.2.1; contained within Genbank accession CP003990); Hyaluronidasefrom bovine testes (EC 3.2.1.35); chondroitin ABC lyase from Proteusvulgaris (EC 4.2.2.4) and heparinase III from Flavobacterium heparinum(EC 4.2.2.8; Genbank accession L12534, preferably version L12534.1).Enzymes for use in the present invention are available from commercialsources, for example Sigma Aldritch.

By “degrade” or “degradation enzyme” means an enzyme which is capable ofbreaking down a protein or carbohydrate. A protein can be broken intopeptide sequences or amino acids, for example by hydrolysis of thepeptide bond. A carbohydrate may be broken down into oligosaccharides orsingle sugar units. A protein and/or carbohydrate may be fully orpartially degraded, meaning that a portion of it may be broken down intosmaller fragments, whereas the remainder of the protein and/orcarbohydrate may be in its native form. Preferably, a degradedextracellular matrix protein or carbohydrate loses some ability toprovide structural and/or biochemical support to a cell, such that anucleic acid sequence introduced into the vitreous can better access aretinal cell. In particular, a degraded extracellular matrix proteinloses some or all its ability to impede movement of a nucleic acidsequence (e.g. gene delivery vector, such as a viral vector), within thevitreous, and into and across the retina. Any loss in extracellularmatrix function is sufficiently minimal so that it does not have anysignificant adverse effect on the eye or vision.

Herein, reference to an extracellular matrix degradation enzyme includesactive fragments thereof. An active fragment may be a portion or shorterversion of the native enzyme, which retains the ability to function asan extracellular matrix degradation enzyme i.e. it retains the abilityto degrade an extracellular matrix protein or carbohydrate, as definedherein. An active fragment may comprise 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% of the sequence of the native enzyme.

Herein, reference to an enzyme includes one or more enzymes. Thus, theinvention provides for the co-administration of a single enzyme or acombination of two or more enzymes. Preferably, where two or moreenzymes are provided, they are each selected from the group definedabove. Where two or more enzymes are administered, they may be providedin separately, sequentially, or two or more may be provided incombination.

Preferably, two enzymes are administered in combination. Where two ormore separate doses of enzyme are provided, any one or more of these maybe provided in combination with the nucleic acid sequence.

An enzyme for use in the present invention may be derived from anysuitable source. The source may be mammalian or non-mammalian. It may bederived from an animal, plant, bacterial, or archeabacterial source.Where mammalian, it is preferred that it is a human enzyme. It may beisolated or purified from such a source. It may be produced as arecombinant protein. Alternatively, it may be synthetically produced.

The nucleic acid and amino acid sequences of enzymes for use in thepresent invention are known in the art.

Herein, enzymes include fragments and derivatives of native enzymes.Preferably a fragment or derivative shares at least 70%, 75%, 80%, 85%or 90%, at least 91, 92, 93, 94, 95, 96, 97, 98, or at least 99%sequence identity with a native enzyme, over a length of 50%, 60%, 70%,80%, 90%, or at least 95% of the length of a native enzyme.

Sequence identity is determined by comparing the two aligned sequencesover a pre-determined comparison window (which may be 50%, 60%, 70%,80%, 90%, 95%, or 100% of the length of the reference nucleotidesequence or protein), and determining the number of positions at whichidentical residues occur. Typically, this is expressed as a percentage.The measurement of sequence identity of a nucleotide sequences is amethod well known to those skilled in the art, using computerimplemented mathematical algorithms such as ALIGN (Version 2.0), GAP,BESTFIT, BLAST (Altschul et al J. Mol. Biol. 215: 403 (1990)), FASTA andTFASTA (Wisconsin Genetic Software Package Version 8, available fromGenetics Computer Group, Accelrys Inc. San Diego, Calif.), and CLUSTAL(Higgins et al, Gene 73: 237-244 (1998)), using default parameters.

An enzyme for use in the present invention may be provided in dry form,which includes either dehydrated or lyophilised forms. Typically, anenzyme will be provided in lyophilised form. Alternatively, an enzymemay be provided as an aqueous solution, for example pre-dissolved inwater at a predetermined concentration and volume. For administration,an aqueous form is preferred, although it is envisaged that a product orkit of the invention may suit the provision of a dried form of theenzyme, optionally with instructions for dissolving. Thus, a method ofthe invention may comprise using a dried enzyme to produce an enzymesolution. Preferably, this is achieved by dissolving or reconstitutingthe enzyme in an aqueous or non-aqueous solvent. Suitable solvents arethose which are non-toxic, and suitable for use with humans or animals.Preferably, a suitable solvent is sterile. An example of a suitablesolvent is sterile phosphate buffered saline. Methods for dissolvingdried proteins are known in the art.

The present invention provides for the administration of a nucleic acidsequence encoding a photosensitive protein to the retina, in order torestore photoreceptive ability to the retina. A photosensitive proteinis one which reacts to light, by undergoing a chemical or physicalchange. By photoreceptive, means a cell which is photosensitive orcomprises a photosensitive protein. The terms photoreceptive orphotoreceptor and photosensitive may be used interchangeably.

A nucleic acid sequence for use in the invention may encode anyphotosensitive protein. Preferably, the nucleic acid sequence of theinvention encodes a mammalian or non-mammalian photosensitive protein.It may be mammalian, non-mammalian, plant, bacterial, or archeabacterialin origin. Where mammalian, it is preferred that it encodes a humanprotein. A nucleic acid sequence for use in the present invention may beselected from the group consisting of rhodopsin, melanopsin, a coneopsin (in particular LWS opsin, MW opsin, and SWS opsin), neuropsin(Opn5), encaphalopsin (Opn3), a parapineal opsin, VAopsin, parapinopsin;parietopsin, pinopsin, TMT opsin, Jelly fish opsin, C-opsin,cryptochrome, and any invertebrate retinal opsins and/or opsins normallysupporting extra-retinal photosensitivity in animals.

A nucleic acid sequence for use in the present invention may be selecteddepending upon the subject to be treated, such that the nucleic acidsequence encodes a photosensitive protein which is native to the retinaof the subject to be treated. Thus, for example, where the subject is ahuman, a nucleic acid sequence will preferably encode a humanphotosensitive protein, for example rhodopsin. However, it is envisagedthat in certain embodiments, a nucleic acid sequence may be providedwhich encodes a photosensitive protein which is not native to thesubject to be treated, but which preferably does not raise an immuneresponse in the subject.

The nucleic acid sequences and amino acid sequences of manyphotosensitive proteins are known in the art. For example, the nucleicacid sequences of preferred photosensitive proteins are provided asfollows:

Melanopsin: Homo sapiens opsin 4 (OPN4), mRNA (cDNA clone MGC:142118IMAGE:8322610), GenBank: BC113558, Version BC113558.1;

Rhodopsin: Homo sapiens rhodopsin (RHO), GenBank: BC111451.3, AccessionNM_000539, Version NM_000539.3 GI:169808383; Cone Homo sapiens opsin 1:Homo sapiens opsin 1, long-wave sensitive, OPN1LW-NCBI ReferenceSequence: Accession: NM_020061, Version NM_020061.5; Homo sapiens opsin1, medium-wave sensitive OPN1MW-NM_000513, version NM_000513.2;

Homo sapiens opsin 1 short-wave-sensitive (OPN1SW) NM_001708, versionNM_001708.2.

Parapinopsin (Genbank Accession NM_001200073, Version NM_001200073.1GI:318056020);

Parietopsin (Genbank Accession DQ100320, Version DQ100320.1GI:73666459); Pinopsin (Genbank Accession AF487546, Version AF487546.1GI:20805654); VA opsin (Genbank Accession AF233520, Version AF233520.1GI:8272567); TMT opsin (Genbank Accessions AH011520 AF349943 AF349944AF349945, version AH011520.2 GI:339511123);

Jelly fish opsin (Genbank Accession AB435549, Version AB435549.1GI:210049957); OPN3 (Genbank Accession NM_014322, Version NM_014322.2GI:71999130); OPN5 (Genbank Accession AY377391, Version AY377391.1GI:38482095); C-opsin (Genbank Accession HF566407, version HF566407.1GI:543581059); and Cryptochrome (Genbank Accession NM_169852, VersionNM_169852.1 GI:24648151).

In the fourth or fifth aspects of the invention described above, thephotoreceptor protein is a human photoreceptor protein. A humanphotoreceptor protein may be human Rhodopsin (also referred to as Rh1,OPN2, RHO) or a photopsin. A photospin may be selected from the groupconsisting of Long Wavelength Sensitive (OPN1LW) Opsin, MiddleWavelength Sensitive (OPN1MW) Opsin and Short Wavelength Sensitive(OPN1SW) Opsin.

Long Wavelength Sensitive (OPN1LW) Opsin has a λ_(max) of 560 nm, in theyellow-green region of the electromagnetic spectrum. It is also referredto as “red opsin”, “L opsin” or “LWS opsin”. Middle Wavelength Sensitive(OPN1MW) Opsin has a λ_(max) of 530 nm, in the green region of theelectromagnetic spectrum. It is also referred to as the “green opsin”,“M opsin” or “MWS opsin”. Short Wavelength Sensitive (OPN1SW) Opsin hasa Amax of 430 nm, in the blue region of the electromagnetic spectrum. Itis also referred to as the “blue opsin”, “S opsin” or “SWS opsin”.

The nucleic acid sequence encoding a human photoreceptor protein may bethe Homo sapiens rhodopsin (RHO) gene (GenBank: BC111451.3, AccessionNM_000539, Version NM_000539.3 GI:169808383), or a fragment orderivative thereof.

The nucleic acid sequence encoding a human photoreceptor protein may bethe Cone Homo sapiens opsin 1, long wave sensitive OPN1LW (NCBIReference Sequence: Accession: NM_020061, Version NM_020061.5), or afragment or derivative thereof.

The nucleic acid sequence encoding a human photoreceptor protein may bethe Cone Homo sapiens opsin 1: medium-wave sensitive OPN1MW, (NCBIReference Sequence: Accession: NM_000513.2; (Science 232 (4747), 193-202(1986)), or a fragment or derivative thereof.

The nucleic acid sequence encoding a human photoreceptor protein may bethe Cone Homo sapiens opsin 1: short-wave-sensitive (OPN1SW) NM_001708,version NM_001708.2, or a fragment or derivative thereof.

Reference to a nucleic acid sequence encoding a photosensitive proteinincludes nucleic acid sequences which are derivatives of the sequencesdescribed herein, or encode a shorter version, or a fragment of aphotosensitive protein, wherein the derivative or fragment retainssubstantially the same photosensitive function as the nativephotosensitive protein. By substantially the same is meant at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the photosensitivefunction of the native protein. A fragment may comprise 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence of the nativeprotein.

Preferably a fragment or derivative of a nucleic acid sequence shares atleast 70%, 75%, 80%, 85% or 90%, at least 91, 92, 93, 94, 95, 96, 97,98, or at least 99% sequence identity with a reference nucleic acidsequence, over a length of 50%, 60%, 70%, 80%, 90%, or at least 95% ofthe length of a reference nucleic acid sequence. A derivative ispreferably active, and may include substitutions and/or deletions and/oradditions compared to the native sequence. Derivatives may also includeportions of other gene sequences, which provide a desired activity orfunction to the photosensitive protein.

Sequence identity is determined by comparing the two aligned sequencesover a pre-determined comparison window (which may be 50%, 60%, 70%,80%, 90%, 95%, or 100% of the length of the reference nucleotidesequence or protein), and determining the number of positions at whichidentical residues occur. Typically, this is expressed as a percentage.The measurement of sequence identity of a nucleotide sequences is amethod well known to those skilled in the art, using computerimplemented mathematical algorithms such as ALIGN (Version 2.0), GAP,BESTFIT, BLAST (Altschul et al J. Mol. Biol. 215: 403 (1990)), FASTA andTFASTA (Wisconsin Genetic Software Package Version 8, available fromGenetics Computer Group, Accelrys Inc. San Diego, Calif.), and CLUSTAL(Higgins et al, Gene 73: 237-244 (1998)), using default parameters.

A nucleic acid sequence may be a DNA, RNA, cDNA, or PNA. It may begenomic, recombinant or synthetic. A nucleic acid sequence may beisolated or purified. It may be single stranded or double stranded.Preferably, a nucleic acid sequence will encode a photosensitiveprotein, as described herein. A nucleic acid sequence may be derived bycloning, for example using standard molecular cloning techniquesincluding restriction digestion, ligation, gel electrophoresis, forexample as described in Sambrook et al; Molecular Cloning: A laboratorymanual, Cold Spring Harbour laboratory Press). A nucleic acid sequencemay be isolated, for example using PCR technology. Such technology mayemploy primers based upon the sequence of the nucleic acid sequence tobe amplified. By isolated is meant that the nucleic acid sequence isseparated from any impurities and from other nucleic acid sequencesand/or proteins which are naturally found associated with the nucleicacid sequence in its source. Therefore, it may be separated fromflanking nucleic acid sequences, or from chromosomal material orsequence. Preferably, it will also be free of cellular material, culturemedium, or other chemicals from a purification/production process. Anucleic acid sequence may be synthetic, for example produced by directchemical synthesis e.g. using the phosphotriester method (Narang et alMeth Enzymol 68: 109-151 1979). A nucleic acid sequence may be providedas naked nucleic acid, or may be provided complexed with a protein orlipid.

The sequence may be altered to improve expression efficiency (forexample by truncating C-terminus or introducing targeting motifs), or toalter characteristics of the light response (for example by removing oradding residues targeted by rhodopsin kinases as part of the signaltermination process).

With the sequence information provided, the skilled person can useavailable cloning techniques to produce a nucleic acid sequence orvector suitable for transduction into a cell.

Preferably, a nucleic acid sequence encoding a photosensitive protein isprovided as a vector, preferably an expression vector. Preferably, itmay be provided as a gene therapy vector, preferably which is suitablefor transduction and expression in a target retinal cell. A vector maybe viral or non-viral (e.g. a plasmid). Viral vectors include thosederived from adenovirus, adenoassociated virus (AAV) including mutatedforms, retrovirus, lentivirus, herpes virus, vaccinia virus, MMLV, GaLV,Simian Immune Deficiency Virus (SIV), HIV, pox virus, and SV40. A viralvector is preferably replication defective, although it is envisagedthat it may be replication deficient, replication competent orconditional. A viral vector may typically persist in an extrachromosomalstate without integrating into the genome of the target retinal cell. Apreferred viral vector for introduction of a nucleic acid sequenceencoding a photosensitive protein to a retinal target cell is an AAVvector, for example self-complementary adenoassociated virus (scAAV).Selective targeting may be achieved using a specific AAV serotype (AAVserotype 2 to AAV serotype 12) or a modified version of any of theseserotypes including AAV 4YF and AAV 7 m8 vectors. In aspects of theinvention where the vector is provided by intra-vitreous administration,the vector may be one which has been modified such that it does not bindto one or more proteins of the ECM. For example, a preferred vector maycomprise a modified heparin sulphate binding site, such that it hasreduced or an inability to bind heperan sulphate, such as AAV 7m8(Dalkara D et al Sci Transl Med 2013; 5:189ra76).

A viral vector may be modified to delete any non-essential sequences.For example, in AAV the virus may be modified to delete all or part ofIX gene, E1a and/or E1b gene. For wild type AAV, replication is atextremely low efficiency, without the presence of helper virus, such asadenovirus. For recombinant adeno-associated virus, preferably thereplication and capsid genes are provided in trans (in pRep/Capplasmid), and only the 2 ITRs of AAV genome are left and packaged into avirion, while the adenovirus genes required are provided either providedby adenovirus or another plasmid. Similar modifications may be made to alentiviral vector.

A viral vector has the ability to enter a cell. However, a non-viralvector such as plasmid may be complexed with an agent to facilitate itsuptake by a target cell. Such agents include polycationic agents.Alternatively, a delivery system such as a liposome based deliverysystem may be used.

A vector for use in the present invention is preferably suitable for usein vivo or in vitro, and is preferably suitable for use in a human.

A vector will preferably comprise one or more regulatory sequences todirect expression of the nucleic acid sequence in a target retinal cell.A regulatory sequence may include a promoter operably linked to thenucleic acid sequence, an enhancer, a transcription termination signal,a polyadenylyation sequence, an origin of replication, a nucleic acidrestriction site, and a homologous recombination site. A vector may alsoinclude a selectable marker, for example to determine expression of thevector in a growth system (for example a bacterial cell) or in a targetretinal cell.

By “operably linked” means that the nucleic acid sequence isfunctionally associated with the sequence to which it is operablylinked, such that they are linked in a manner such that they affect theexpression or function of one another. For example, a nucleic acidsequence operably linked to a promoter will have an expression patterninfluenced by the promoter.

A promoter mediates expression of the nucleic acid sequence to which itis linked. A promoter may be constitutive or may be inducible. Apromoter may direct ubiquitous expression in the inner retinal cells, orneurone specific expression. In the latter case, a promoter may directcell type specific expression, for example to ON bipolar or OFF bipolarcells. Suitable promoters will be known to persons skilled in the art.For example, a suitable promoter may be selected from the groupconsisting of L7, thy-1, recoverin, calbindin, human CMV, GAD-67,chicken beta-actin, hSyn, Grm6, Grm6 enhancer-SV40 fusion protein.Targeting may be achieved using cell specific promoters, for examplee.g. Grm6-SV40 for selective targeting of ON-bipolar cells. The Grm6promoter is a fusion of 200-base pair enhancer sequence of the Grm6 geneencoding for ON-bipolar cell specific metabotropic glutamate receptor,mGluR6, and an SV40 eukaryotic promoter. Preferred sources of the Grm6gene are mouse and human. Ubiquitous expression may be achieved using apan-neuronal promoter, examples of which are known and available in theart. One such example is CAG. The CAG promoter is a fusion of CMV earlyenhancer and chicken β-actin promoter.

The present invention provides a therapeutic composition comprising i) anucleic acid sequence encoding a photosensitive protein and ii) anextracellular matrix degradation enzyme. A composition may be providedin a pharmaceutically acceptable excipient.

The present invention also provides a nucleic acid vector comprising anucleic acid encoding a human photoreceptor protein. The humanphotoreceptor protein may be human Rhodopsin (also referred to as Rh1,OPN2, RHO) or a photopsin. A photospin may be selected from the groupconsisting of Long Wavelength Sensitive (OPN1LW) Opsin, MiddleWavelength Sensitive (OPN1MW) Opsin and Short Wavelength Sensitive(OPN1SW) Opsin. The vector may be provided as a composition, foradministration to a subject.

A composition may be a liquid or a solid, for example a powder, gel, orpaste. Preferably, a composition is a liquid, preferably an injectableliquid. Suitable excipients will be known to persons skilled in the art.

In the fourth and fifth aspects of the invention relating to theadministration of a nucleic acid vector comprising a nucleic acidencoding a human photoreceptor protein, the vector may be administeredto an eye by sub-retinal or intra-vitreous administration. In eithermode of administration, the vector is preferably provided as aninjectable liquid. Preferably, the injectable liquid is provided as acapsule or syringe. In this aspect, preferably the nucleic acid isintroduced without administration of an extracellular matrix degradationenzyme (i.e. it is introduced without co-administration of an enzyme,wherein co-administration includes separate, sequential or combinedadministration during the same therapy) as defined herein. In thisaspect, preferably the injectable liquid does not comprise anextracellular matrix degradation enzyme

In aspects of the invention comprising administration of anextracellular matrix degradation enzyme, an enzyme may be providedseparately or in combination with the nucleic acid sequence, i.e. as asingle composition. Where provided separately, the enzyme and nucleicacid sequence may be provided in the same excipient or in differentexcipients. In such an embodiment, the may be held separately, forexample in separate microcapsules. Thus, in a preferred embodiment, thepresent invention provides a composition comprising i) a firstinjectable liquid comprising a nucleic acid sequence encoding aphotosensitive protein; and ii) a second injectable liquid comprising anextracellular matrix degradation enzyme. Preferably, the first andsecond injectable liquids are provided in separate containers, such ascapsules or syringes, preferably within the same packaging.

Preferably, a composition of the first, second and third aspects of theinvention is provided for separate, sequential or combinedadministration of the nucleic acid and enzyme to a subject

A composition of the invention may be provided for use in a method ofproviding photoreceptor function to a cell. In an embodiment, acomposition of the invention may be provided for use in a method ofrestoring photoreceptor function of a retina. In an embodiment, acomposition of the invention may be provided for use in a method ofrestoring vision to a subject. In an embodiment, a composition of theinvention may be provided for use in a method of treating a retinaldegenerative condition, for example a retinal dystrophy including a roddystrophy, a rod-cone dystrophy, a cone-rod dystrophy, a cone dystrophyand a macular dystrophy; other forms of retinal or macular degeneration,an ischaemic condition, uveitis and any other disease resulting fromloss of photoreceptor ability, e.g retinal pigmentosa.

The present invention provides a kit comprising a nucleic acid vectorcomprising a nucleic acid encoding a human photoreceptor protein. Thehuman photoreceptor protein may be as described above. The nucleic acidvector may be provided as a composition, as described herein. Thecomposition may be an injectable liquid. In this aspect, preferably thekit does not comprise an extracellular matrix degradation enzyme.

The present invention provide a kit comprising i) a nucleic acidsequence encoding a photosensitive protein; and ii) an extracellularmatrix degradation enzyme. In a kit of the invention, the extracellularmatrix degradation enzyme and nucleic acid sequence may be providedseparately, or in combination. They may each, independently, be providedas a composition, for example as described herein. Thus, a kit of theinvention may comprise i) a nucleic acid sequence encoding aphotosensitive protein and ii) an extracellular matrix degradationenzyme, wherein the nucleic acid sequence and/or the enzyme are providedas a composition. The nucleic acid sequence and enzyme may be providedin combination, in a single composition, or may be provided as separatecompositions. Where provided separately, the enzyme and nucleic acidsequence may be provided in the same excipient or in differentexcipients. In such an embodiment, the may be held separately, forexample in separate microcapsules.

In a preferred embodiment, a kit may comprise i) a first injectableliquid comprising a nucleic acid sequence encoding a photosensitiveprotein and ii) a second injectable liquid comprising an extracellularmatrix degradation enzyme. Preferably, the first and second injectableliquids are provided in separate containers, such as capsules orsyringes, preferably within the same packaging.

A kit of the invention may further comprise instructions for use, adosage regimen, one or more fine needles, one or more syringes, andsolvent.

A kit of the invention may be provided for use in a method of providingphotoreceptor function to a cell. In an embodiment, a kit of theinvention may be provided for use in a method of restoring photoreceptorfunction of a retina. In an embodiment, a kit of the invention may beprovided for use in a method of restoring vision to a subject. In anembodiment, a kit of the invention may be provided for use in a methodof treating a retinal degenerative condition, for example a retinaldystrophy including a rod dystrophy, a rod-cone dystrophy, a cone-roddystrophy, a cone dystrophy and a macular dystrophy; other forms ofretinal or macular degeneration, an ischaemic condition, uveitis and anyother disease resulting from loss of photoreceptor ability.

The present invention provides a method of providing photoreceptorfunction to a cell, the method comprising introducing into an eye anucleic acid vector comprising a nucleic acid encoding a humanphotoreceptor protein. The human photoreceptor protein may be asdescribed above. The method may comprise sub-retinal or intra-vitreousadministration of the nucleic acid vector to the inner retinal cells ofthe eye. In this aspect, preferably the method does not compriseadministration of an extracellular matrix degradation enzyme. Thepresent invention provides a nucleic acid vector comprising a nucleicacid encoding a human photoreceptor protein for use in a method oftreating retinal degeneration by providing photoreceptor function to acell. In this aspect, preferably an extracellular matrix degradationenzyme is not used (i.e. the nucleic acid is introduced withoutco-administration of an enzyme, wherein co-administration includesseparate, sequential or combined administration during the sametherapy).

The present invention provides a method of providing photoreceptorfunction to a cell, the method comprising introducing into the vitrealcavity of an eye i) a nucleic acid sequence encoding a photosensitiveprotein and ii) an extracellular matrix degradation enzyme. The presentinvention provides i) a nucleic acid sequence encoding a photosensitiveprotein and ii) an extracellular matrix degradation enzyme for use in amethod of providing photoreceptor function to a cell.

The present invention also provides a method of augmenting photoreceptorfunction in a retina, in particular following rod and/or cone celldegeneration, the method comprising introducing into the vitreal cavityof an eye a nucleic acid vector comprising a nucleic acid encoding ahuman photoreceptor protein. The human photoreceptor protein may be asdescribed above. The method may comprise sub-retinal or intra-vitreousadministration of the nucleic acid vector to the inner retinal cells ofthe eye. In this aspect, preferably the method does not compriseadministration of an extracellular matrix degradation enzyme. Thepresent invention provides a nucleic acid nucleic acid vector comprisinga nucleic acid encoding a human photoreceptor protein for use intreating retinal degeneration by augmenting photoreceptor function in aretina. The human photoreceptor protein may be as described above. Inthis aspect, preferably an extracellular matrix degradation enzyme isnot used (i.e. the nucleic acid is introduced without co-administrationof an enzyme, wherein co-administration includes separate, sequential orcombined administration during the same therapy).

The present invention also provides a method of augmenting photoreceptorfunction in a retina, in particular following rod and/or cone celldegeneration, the method comprising providing photoreceptor function toa cell as described herein. The present invention provides i) a nucleicacid sequence encoding a photosensitive protein and ii) an extracellularmatrix degradation enzyme for use in a method of augmentingphotoreceptor function in a retina, in particular following rod and/orcone cell degeneration, wherein the method comprises providingphotoreceptor function to a cell as described herein.

The present invention also provides a method of restoring vision to asubject, the method comprising introducing into an eye a nucleic acidvector comprising a nucleic acid encoding a human photoreceptor protein.The human photoreceptor protein may be as described above. The methodmay comprise sub-retinal or intra-vitreous administration of the nucleicacid vector to the inner retinal cells of the eye. In this aspect,preferably the method does not comprise administration of anextracellular matrix degradation enzyme. The present invention providesa nucleic acid nucleic acid vector comprising a nucleic acid encoding ahuman photoreceptor protein for use in restoring vision to a subject.The human photoreceptor protein may be as described above. In thisaspect, preferably an extracellular matrix degradation enzyme is notused (i.e. the nucleic acid is introduced without co-administration ofan enzyme, wherein co-administration includes separate, sequential orcombined administration during the same therapy).

The present invention also provides a method of restoring vision to asubject, the method comprising providing photoreceptor function to acell as described herein. The present invention provides i) a nucleicacid sequence encoding a photosensitive protein and ii) an extracellularmatrix degradation enzyme for use in a method of restoring vision to asubject, wherein the method comprises providing photoreceptor functionto a cell as described herein.

The present invention also provides a method of treating retinal diseasea subject, the method comprising introducing into an eye a nucleic acidvector comprising a nucleic acid encoding a human photoreceptor protein.The human photoreceptor protein may be as described above. The methodmay comprise sub-retinal or intra-vitreous administration of the nucleicacid vector to the inner retinal cells of the eye. In this aspect,preferably the method does not comprise administration of anextracellular matrix degradation enzyme. The present invention providesa nucleic acid vector comprising a nucleic acid encoding a humanphotoreceptor protein for use in treating retinal disease in a subject.The human photoreceptor protein may be as described above. In thisaspect, preferably an extracellular matrix degradation enzyme is notused (i.e. the nucleic acid is introduced without co-administration ofan enzyme, wherein co-administration includes separate, sequential orcombined administration during the same therapy). The disease may be aretinal dystrophy including a rod dystrophy, a rod-cone dystrophy, acone-rod dystrophy, a cone dystrophy and a macular dystrophy; otherforms of retinal or macular degeneration, an ischaemic condition,uveitis and any other disease resulting from loss of photoreceptorability.

The present invention also provides a method of treating a retinaldegenerative disease, the method comprising providing photoreceptorfunction to a cell as described herein. Thus, the present inventionprovides i) a nucleic acid sequence encoding a photosensitive proteinand ii) an extracellular matrix degradation enzyme for use in a methodof treatment of a disease, as defined herein. The disease may be aretinal dystrophy including a rod dystrophy, a rod-cone dystrophy, acone-rod dystrophy, a cone dystrophy and a macular dystrophy; otherforms of retinal or macular degeneration, an ischaemic condition,uveitis and any other disease resulting from loss of photoreceptorability.

By providing photoreceptor function to a cell means that a cell whichpreviously did not have photoreceptor ability or whose photoreceptorability has degenerated, wholly or partially, becomes photo-receptiveupon expression therein of the foreign nucleic acid sequence encoding aphotosensitive protein. Such a cell may be referred to herein as atransformed cell, because it comprises therein non-native nucleic acid.Preferably, a transformed retinal cell exhibits some or all of thephotoreceptor ability of a native photoreceptive cell. Preferably, atransformed cell exhibits at least the same or substantially the samephotoreceptive ability of a native retinal photoreceptor cell.

Preferably, a transformed cell exhibits higher photoreceptive abilitythan a diseased or degenerating native retinal photoreceptor cell.Therefore, a transformed cell will preferably have increasedphotoreceptor compared to a degenerated or diseased cell from the samesource, maintained under the same conditions, without treatment. Atransformed cell can be distinguished from a native cell by the presencetherein of exogenous nucleic acid.

By augmenting photoreceptor function is meant increasing photoreceptorfunction of the retina, either by increasing the function inphotoreceptor cells such as rod or cone cells, and/or by providingphotoreceptor function to a cell. Thus, the retina will have anincreased ability to receive light signals and transmit such signalscompared to a retina which has not been treated with method as describedherein. The increase may be by any amount, preferably to wild typelevels.

By restoring vision in a subject is meant that the subject showsimproved vision compared to prior to treatment, for example using visiontests as described herein. Restoring includes any degree in improvement,including full restoration of vision to perfect or near perfect vision.

By treating disease is meant administration of as nucleic acid andextracellular degradation enzyme as described herein to ameliorate oralleviate of one or more symptoms of a disease selected from the groupconsisting of a retinal dystrophy including a rod dystrophy, a rod-conedystrophy, a cone-rod dystrophy, a cone dystrophy and a maculardystrophy; another forms of retinal or macular degeneration, anischaemic conditions, uveitis and any other disease resulting from lossof photoreceptor ability. Amelioration or alleviation may result in animprovement of peripheral or central vision, and/or day or night vision.

Methods of the invention comprise introducing into the vitreal cavity ofan eye a nucleic acid sequence encoding a photosensitive protein.Preferably, the method comprises contacting a cell with a nucleic acidsequence encoding a photosensitive protein. Preferably, a cell is aretinal cell, preferably an ON-bipolar cell, an OFF-bipolar cell, ahorizontal cell, a ganglion cell and/or an amacrine cell.

Preferably, a method of the invention comprises targeting a nucleic acidsequence encoding a photosensitive protein to the retina of an eye,preferably to a non-photoreceptive cell of the retina, preferably to anON-bipolar cell, an OFF-bipolar cell, a horizontal cell, a ganglion celland/or an amacrine cell. Thus, by contacting a cell includestransfection and/or transduction of a cell.

Where an enzyme is administered, the enzyme does not need to beinternalised into a retinal cell, but may remain in the vitreal cavityor in the retina, where it degrades extracellular matrix proteins toimprove access of the nucleic acid sequence to the retinal cells.

A method of the invention is preferably performed in vivo.

The nucleic acid sequence encoding a photosensitive protein and anenzyme may be provided separately or sequentially or in combination.Where provided simultaneously (i.e. in combination), a nucleic acidsequence and an enzyme may be provided as a single composition which isintroduced into the vitreal cavity, or may be provided as separatecompositions but provided to the vitreal cavity simultaneously. Ifprovided separately, a nucleic acid sequence and enzyme may be providedin separate compositions, and may be provided either at the same time,or sequentially. When provided sequentially, an enzyme may be providedbefore or after a nucleic acid sequence, preferably before. In thefourth and fifth aspects, an enzyme is not con-administered by themethods described above.

Where two or more enzymes are provided, they may be introduced in acombined single dose or in multiple doses. An enzyme dose may beprovided in combination with a nucleic acid sequence, or separatelythereto. Where multiple enzyme doses are introduced, any one or moredoses may be a combined enzyme/nucleic acid sequence dose. A preferredmethod comprises the introduction of i) a combined enzyme dose, and ii)a sequential dose of nucleic acid sequence. In a preferred embodiment,the enzyme dose comprises heparinase III and hyaluronan lyase.

Any suitable method may be used for introducing a nucleic acid sequenceand enzyme to the sub-retina or vitreal cavity. A preferred method isinjection. Thus, a dose of nucleic acid sequence and/or enzyme may beprovided as an injection. A method of the invention may compriseinjecting sub-retinally or into the vitreal cavity a nucleic acid vectorcomprising a nucleic acid sequence encoding a human photoreceptorprotein. Preferably, the method is to provide a photoreceptor functionto a cell, for example to restore vision, preferably for treatment of aretinal degenerative condition for example a retinal dystrophy includinga rod dystrophy, a rod-cone dystrophy, a cone-rod dystrophy, a conedystrophy and a macular dystrophy; another forms of retinal or maculardegeneration, an ischaemic conditions, uveitis and any other diseaseresulting from loss of photoreceptor ability.

A method may comprise injecting into the vitreal cavity, separately,simultaneously or sequentially, i) a nucleic acid sequence encoding aphotosensitive protein and ii) an extracellular matrix degradationenzyme. In a preferred embodiment, a method of the invention comprisesinjecting a single dose comprising a i) nucleic acid sequence encoding aphotosensitive protein and ii) an extracellular matrix degradationenzyme into the vitreal cavity of an eye. Preferably, a method comprisesinjecting a single dose comprising i) a nucleic acid sequence encodingrhodopsin; and ii) the enzymes heparinase III and hyaluronan lyase, intothe vitreal cavity of an eye. In a preferred embodiment, the inventionprovides a single injectable dose comprising i) a nucleic acid sequenceencoding a photosensitive protein and ii) an extracellular matrixdegradation enzyme for introduction into the vitreal cavity of an eye toprovide a photoreceptor function to a cell, for example to restorevision, preferably for treatment of a retinal degenerative condition forexample a retinal dystrophy including a rod dystrophy, a rod-conedystrophy, a cone-rod dystrophy, a cone dystrophy and a maculardystrophy; another forms of retinal or macular degeneration, anischaemic conditions, uveitis and any other disease resulting from lossof photoreceptor ability. Preferably, the invention provides a singleinjectable dose comprising i) a nucleic acid sequence encodingrhodopsin; and ii) the enzymes heparinase III and hyaluronan lyase, forintroduction into the vitreal cavity of an eye to provide aphotoreceptor function to a cell, for example to restore vision,preferably for treatment of a retinal degenerative condition for examplea retinal dystrophy including a rod dystrophy, a rod-cone dystrophy, acone-rod dystrophy, a cone dystrophy and a macular dystrophy; anotherforms of retinal or macular degeneration, an ischaemic condition,uveitis and any other disease resulting from loss of photoreceptorability.

Where a nucleic acid sequence and one or more enzymes are provided inmultiple (two or more) doses, these may be separated by suitable timeintervals, for example 30 seconds to several hours or 1 or more days.

Each dose may comprise an effective amount of a nucleic acid sequenceand/or an enzyme. An effective dose of a nucleic acid sequence may rangefrom 1×10⁹ to 1×10¹⁴ or 7.5×10¹⁵, preferably 1×10¹¹ to 7.5×10¹³ nucleicacid sequences per treatment regimen (e.g. number of vectors or virusparticles). An enzyme may be provided at a dose of 0.075-0.125 units pereye, or more.

A method of the invention may comprise a step of diagnosing a subjectfor a retinal degenerative condition, for example a retinal dystrophyincluding a rod dystrophy, a rod-cone dystrophy, a cone-rod dystrophy, acone dystrophy, a macular dystrophy; another forms of retinal or maculardegeneration, an ischaemic conditions, uveitis and any other diseaseresulting from loss of photoreceptor ability. A diagnostic step maycomprise a visual test, for example a pupillary light reflex (PLR) test,visual acuity test (LogMAR), clinical diagnostic tests for examplebiomicroscopy/slit-lamp ocular/retinal clinical examination; colourvision testing, visual field testing, contrast/full field sensitivity;electrodiagnostic tests including for example EGGs, VEPs; imaging,retinal fundus photography, OCT, and adaptive optics scanning laserophthalmoscope (AOSLO). Other suitable tests will be known to personsskilled in the art.

A method of the invention may comprise a step of dilating the pupil ofan eye to be treated, for example by application of mydriatic, forexample tropicamide and/or phenylephrine and/or cyclopentolate. A methodof the invention may comprise a step of accessing the retina, forexample by surgery.

A method of the invention may comprise monitoring the vision of asubject who has been treated for any improvement in vision. Improvementsin vision may be any one or more of the following: increased pupillarylight reflex (PLR), increased contrast sensitivity, increased resolutionof low or high frequency flicker, and increased detection of movingimages. In addition, increased light induced locomotor activity may beimproved in animals such as mice. An improvement in vision may be anability to respond to or detect light at 10¹⁵-10¹³ photon/cm²/s cornealirradiance. An improvement in vision may comprise an ON-sustained,ON-transient, OFF-excitatory, OFF-inhibitory or ON-OFF response.Preferably, monitoring improvement may comprise a method of quantifyingthe subjects subjective visual experience or an objective measure oflight response, for example a pupillary light reflex (PLR) test, LogMARvisual acuity, clinical examination slit-lamp biomicroscopy; colourvision testing, visual field testing, contrast/full field sensitivity;electrodiagnostics—ERGs, VEPs; imaging: retinal fundus photography, OCT,adaptive optics scanning laser ophthalmoscope (AOSLO), or mazenavigation tasks.

A subject may be monitored every 6, 8, 10, 12 or 24 hours, or every 2,3, 4, 5 days. This may be repeated after 1, 2, 3, 4, 5, 6 months or ayear or more.

The term “in vivo” refers to the natural environment (e.g., in an animalor a cell) and to processes or reaction that occur within that naturalenvironment (for example on the body of a subject).

The present invention is based upon targeting a nucleic acid sequenceencoding a photosensitive protein to retinal cells, to compensate fordegeneration of photoreceptor cells in the retina. The cells to whichthe nucleic acid sequence is targeted are cells of the retina which arealive and capable of expressing a foreign nucleic acid sequence. Herein,a retinal cell is a cell of the retina, which is a nerve or neuron celland is capable of becoming excited and transmitting an electricalsignal. Preferably, a target retinal cell will be capable of generatingan electrical signal and initiating the signalling cascade leading totransmission of signal to the optic nerve. Preferably, the targetretinal cells are cells of the inner retina. A target cell may be a rodor cone cell, and/or may be a non-photoreceptor cell (i.e. a retinalcell which in its native form does not respond to light). A targetretinal cell may include one or more cell types be selected from thegroup consisting of rod cells, cone cells, ON-bipolar cells, OFF-bipolarcells, horizontal cells, ganglion cells, Muller cells and/or amacrinecells.

Thus, where a target retinal cell is targeted to an ON-bipolar cell,OFF-bipolar cell, horizontal cell, ganglion cell and/or amacrine cell ofthe retina, the expression of the nucleic acid encoding a photosensitiveprotein may be referred to as ectopic expression. Thus, the presentinvention includes within its scope a method of ectopically expressing anucleic acid sequence encoding a photosensitive protein in anon-photoreceptor cell. Such ectopic expression has the effect ofproviding photoreceptor function to a cell, by expression of aheterologous photosensitive protein therein. This serves to increase thephotoreceptive capacity of the retina where degeneration is observed.The co-administration of extracellular matrix degradation enzymes withthe nucleic acid sequence serves to improve transduction of the nucleicacid sequence into the target retinal cells.

A cell may be a prokaryotic or eukaryotic. It may be a bacterial cellsuch as E. coli, or may be a mammalian or non-mammalian cell, forexample an insect cell, a yeast cell, a cell line or a cell freeexpression systems, for example for use in generating a vector orcomposition of the invention. A target retinal cell may be an ON-bipolarcell, an OFF-bipolar cell, a horizontal cell, a ganglion cell and/or anamacrine cell.

Horizontal cells are inner retina cells, involved in signal processingand feedback to photoreceptor cells; bipolar cells are inner retinalcells and communicate between rods/cone cells and the amacrine and/organglion cells; amacrine cells are found in inner retina and allowcommunication between photoreceptor pathway and ganglion cells; ganglioncells are innermost retinal cells which pass signal from photoreceptorcells to the optic nerve.

Reference to a cell herein includes progeny of the cell. Preferably,modifications to a cell according to the present invention also occur insucceeding generations of the transformed host cells. Progeny cellswhich may not be identical to the initial targeted cell but preferablywill also exhibit expression of the non-native photosensitive protein.

The present invention may be used for the treatment of any disorderwhich is characterised by a degeneration of photoreceptor cells in theeye, typically a degeneration which is sufficient to result in partialor complete loss of vision. Examples of conditions which may be treatedor ameliorated by the present invention include a retinal dystrophy(retinal pigmentosa) including a rod dystrophy, a rod-cone dystrophy, acone-rod dystrophy, a cone dystrophy and a macular dystrophy; otherforms of retinal or macular degeneration (e.g. age related maculardegeneration), an ischaemic condition, edema (macular or retinal),uveitis and any other disease resulting from loss of photoreceptorability.

As used herein, the term “subject” refers to any mammal or non-mammal.Mammals include but are not limited to, humans, vertebrates such asrodents, non-human primates, cows, horses, dogs, cats, pigs, sheep,goats, giraffes, yaks, deer, camels, llamas, antelope, hares, andrabbits.

Herein reference to “a” or “an” includes within its scope both thesingular, and the plural, i.e. one or more.

Unless stated otherwise, the features and embodiments of each aspectapplies to the other aspects of the invention, mutatis mutandis.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean “including but notlimited to”, and they are not intended to (and do not) exclude othermoieties, additives, components, integers or steps. Throughout thedescription and claims of this specification, the singular encompassesthe plural unless the context otherwise requires. In particular, wherethe indefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the contextrequires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith. All of the features disclosed in this specification(including any accompanying claims, abstract and drawings), and/or allof the steps of any method or process so disclosed, may be combined inany combination, except combinations where at least some of suchfeatures and/or steps are mutually exclusive. The invention is notrestricted to the details of any foregoing embodiments. The inventionextends to any novel one, or any novel combination, of the featuresdisclosed in this specification (including any accompanying claims,abstract and drawings), or to any novel one, or any novel combination,of the steps of any method or process so disclosed.

Examples

In this study the feasibility of restoring light responsiveness to blindrd1 retinas by expressing human rod opsin in surviving inner retinalneurons using intravitreal AAV gene therapy was investigated. Bothuntargetted (neuron non-selective) and targetted (selective to ONBPcells) delivery was studied. Furthermore, the properties of restoredresponses in vivo and the ability of treated blind mice to resolve moresophisticated image processing including full field flickers, contrastdetection, and naturalistic scenes were examined. Both untargetted andtargetted ectopic rod opsin successfully recovers vision through adiverse visual code involving both ON and OFF pathways were found.Restored responses are transmitted beyond the retina into CNS visualpathways and function under the light intensity range of normal cone(and rod) vision as well as under light adapted conditions. Theresponses are robust enough to lead to light-induced locomotor behaviourin treated mice under illumination equivalent of indoor room lighting.Furthermore, the results show that mice with specifically targettedON-bipolar cells, are able to resolve more complex visual functionsincluding full field flickers, contrast detection and naturalistic moviescenes featuring changes in spatial patterns and object motion.

1. Intraocular Injections and Gene Delivery Via AAV (FIG. 1)

Adult C3H/HeJ (rd1) mice were used in this study. Specifically, a nullmutation pde65b in rd1 mice leads to a complete loss of photoreceptorsby p90 (Crater-Dawson et al, 1978, Farber db et al 1994). (Important tonote that mutation does not affect rhodopsin itself). In order to answerwhether rod opsin can express outside photoreceptors in vivo, the geneexpression cassette was injected (FIG. 1A) intra-vitreally into adultrd1 mice. Bearing in mind translational potential of this approach aclinically safe AAV2/2 vector was used and a humanised version of rodopsin. Intravitreal injection was employed in order to achieve morewidespread retinal transduction and minimise potential complicationsassociated with an alternative sub-retinal delivery of vectors.Sub-retinal approach normally leads to transduction localised to thesite of injection. An optimised combination of glycosidic enzymes wasco-injected in order to enhance transduction and allow vector topenetrate deeper into the retina. It is unknown which cell types, ifany, outside native photoreceptors would express rod opsin. Untargetedexpression was studied first, using a strong pan-neuronal promoter, CAG.

Retinas were harvested four to six months post injection and retinalcryo-sections were immuno-labelled with antibody against human rodopsin. Expression was confirmed using fluorescent microscopy. Rod opsinexpressed well outside photoreceptors as confirmed by strongly labelledcell somas (FIG. 1B). Expression was localised to the plasma membraneand was observed in both RGC layer (FIG. 1C) and INL (FIG. 1D). It islikely that with a non-selective promoter several different cell typesin the INL were transduced, including some horizontal and amacrinecells, although by large the transduced cell bodies in the INL hadcharacteristics of bipolar cells (FIG. 1D). Rod opsin expression waspan-retinal, albeit in the INL it was patchy with variable depths ofpenetration. In comparison, no fluorescence was seen in our control PBSinjected group. In addition, the un-injected wild type retinas treatedwith antibody against rod opsin showed clear fluorescence inphotoreceptor outer segments indicating that the antibody is specificfor rod opsin with no off-target labelling.

Results:

Pupillometry:

FIG. 2. Restoration of pupillary light reflex following rhodopsintreatment. Irradiance-response curves for maximum pupillary constrictionduring 10 s of white light at neutral density filters ND4 (dimmest) toND0 (brightest). Rhodopsin treated eyes (Rd RHO) show a markedimprovement in visual sensitivity compared to GFP injected eyes (RdGFP). Data for wild-type C57 mice injected with PBS/enzyme mixture areshown for comparison (WT PBS). Data are normalised to pupil sizeimmediately preceding the onset of light. Values are mean±SEM, with nindicating the number of eyes examined.

In-Vivo Electrophysiology:

FIG. 3. Data from in-vivo electrophysiology recordings. Representativesample of peri-event histograms for a number of recording channels(sigs) showing the mean firing rate of LGN cells to 2 s stimulus of 405nm light presented to a contralateral rhodopsin treated eye atincreasing intensities (FIG. 3A—ND2, B-ND1, C-ND0; ND2=dimmest,ND0=brightest). A number of channels (e.g. sig 50, sig 52 at ND0 andND1) showed robust ‘fast-onset’ light responses with an ‘ON peak’ afterlight is switched on, and an ‘OFF peak’ when light is switched off. Theobserved responses are clearly distinct from native light responsesnormally present in rd1 mouse LGN following stimulation of contralateraleye (FIG. 3). These native responses were observed from both rhodopsinor GFP treated eyes and show typical ‘slow-onset’ sustained, responses(from melanopsin retinal ganglion cells, FIG. 3D) and very transientfast responses (from surviving cone photoreceptors, FIG. 3E). The‘restored’ responses (FIG. 3A-C) therefore come from retinal cells(retinal ganglion or bipolar cells) with induced photoreceptiveproperties from ectopic expression of rhodopsin.

MEA In-Vitro Recordings:

FIG. 4. Data from in-vitro MEA recordings. Representative sample ofperi-event histograms for a number of recording channels (sigs) showingthe mean firing rate of retinal ganglion cells to 2 s stimulus of 405 nmlight applied to isolated retinas at increasing intensities (FIG.4A—ND2, B-ND1, C-ND0; ND2=dimmest, ND0=brightest). A number of channelsshowed robust ‘fast-onset’ light responses with some channels (e.g. sig35, sig 36 at ND0 and ND1) showing an ‘ON peak’ after light is switchedon, and an ‘OFF peak’ when light is switched off. The observed responsesare clearly distinct from native light responses normally present in rd1mouse retina following light stimulation (FIG. 5). These nativeresponses were observed from both rhodopsin or GFP treated eyes and showtypical ‘slow-onset’ sustained responses (from melanopsin retinalganglion cells, e.g. FIG. 5, sig 37). The novel responses (FIG. 4A-C)therefore originate from retinal cells with rendered photoreceptiveproperties from ectopic expression of rhodopsin.

Restoring Light-Evoked Activity in Retinal Ganglion Cells—FunctionalEvaluation of Rod Opsin Responses In-Vitro

Next, it was desired to confirm that the ectopically expressed rod opsincould drive functional light responses in the blind retina. Retinalexplants were mounted onto multi-electrode array to test light evokedactivity. Rod opsin injected (CAG-hRho) retinas from rd1 mice (4-6 mold) showed light responsive cells with robust initial firing rate afterstimulation with two-second full-field flashes of broad spectrum whitelight at 15.4 log photons/cm2/s. However, after a few repeatedpresentations, light evoked activity diminished to low frequency ofspiking, suggesting a bleaching effect. This was not surprising forin-vitro preparations where endogenous supplies of 9-cis are quicklydepleted. However, addition of 9-cis to the preparation evoked robustneuronal activity with strongly reproducible responses. In addition,diverse response profiles were noted across the population of lightresponsive cells (n=6 retinas) including ON sustained, ON transient andOFF responses. In contrast, the GFP injected (CAG-GFP) age-matchedcontrol retinas showed no light evoked activity after presentation ofthe same light stimulus consistent with absence of photoreceptors (n=2retinas). (It was also possible to drive light responses in rod opsintreated retinas at lower light intensities (14.4 log photons/cm2/s FIG.1H and 13.4 log photons/cm2/s)) representing a significant improvementin light sensitivity compared to other optogenetic strategies which useat least 5-6 log units higher light intensities than necessary toactivate native cone vision (Gaub, Lagali etc).

Ectopic Rod Opsin Mediated Responses are Transmitted to theLGN—Characterisation of Restored Responses In-Vivo (FIG. 3)

Since rod opsin bleaching, without constant exogenous supplies ofchromophore (9-cis retinal), was a problem for in-vitro experimentsneuronal activity was tested in-vivo in order to determine if therewould be enough retinal in intact retinas to allow the chromophorerecycling and prevent bleaching. In addition, whether or not therestored responses are robust enough to activate higher visual centresin the context of degenerating and remodelling retina was investigated.Neuronal responses from the dorsal lateral geniculate nucleus, dLGN,were recorded from both hemi-spheres simultaneously, where one eye waspreviously treated with rod opsin and the other, injected with GFP,served as an internal control. The dLGN is the major retinorecipientpart of the brain and contains the neurons that relay signals to thevisual cortex. Responses from the dLGN, the first synaptic connectionfor the RGCs, were studied to get the best idea of the properties ofrestored responses. A significantly increased number of light responsesacross the dLGN (157) were found compared to internal controls (4)following two-second full-field flashes of 410 nm light at 15.4 logphotons/cm2/s. Light responsive cells were identified according toobjective criteria using peri-stimulus time histogram (PSTH) analysis inthe Neuroexplorer (Nex Technologies) where average firing rate had tocross 95% CI after stimulus onset or offset, compared to the baseline.PSTH time interval for 2 s stimulus was −2 s to 5 s, and bin size 250ms. Clear artefacts leading to false positives were excluded.‘Heat-maps’ were generated to include all light responsive cells fromeach group and order responses according to the amplitude of thesustained response during the initial 1 second of a 2-second stimuluspresentation. Green colours (+1) represent high firing rates andpink/purple colours (−1) low firing rates. FIG. 3F clearly depicts adiverse nature of restored responses including sustained, transient, ONand OFF responses, as are normally found in WT retinas. In contrast,light responsive cells (n=4) that were found in the control group wereall slow onset ON sustained, likely to originate from native ipRGCs.Bleaching was not found to be a problem for in-vivo light responses,which showed robust firing across many repeated trials.

To capture the diversity of restored responses seen at a single unitlevel (but found across all retinas tested) individual responses werestudied systematically through their peri-stimulus time histograms(PSTH) and corresponding trial bin counts (TBC). Averaging across thepopulation of cells would be difficult given that some cells are excitedby light ON (ON responses) some inhibited by light ON (OFF inhibitoryresponses), some excited by light OFF (OFF excitatory) and some excitedby both light ON and OFF (ON-OFF cells). Both sustained (10 s and 2 slight step, FIG. 3G) and more transient responses (2 s light step, FIG.3H) were evoked in treated eyes, comparable to those from WT retinas(insert). In addition, to ON responses we also found many OFF responses(FIG. 3I) and some ON-OFF responses (FIG. 3J). Across the population oflight responsive cells, 99/157 (63.1%) of ON cells, 46/157 (29.3%) ofOFF cells and 12/157 (7.6%) of ON-OFF cells (FIG. 3E) were found. Thesewere broadly categorised into ON sustained cells (51/157; 32.5%), ONtransient cells (48/157; 30.6%), OFF excitatory cells (29/157; 18.5%),OFF inhibitory cells (17/157; 10.8%) and ON-OFF cells (12/157; 7.6%).Mean firing rate ranged between 20-100 Hz across the restored responses.Responses were found with short latencies (onset within 50-100 ms-2D,cell 1; 2E ce111) as well as longer latencies (onset within 500 ms-2D,cell 3). Firing rate for most sustained responses returned rapidly backto baseline (hundreds of millisecond of turning the light stimulus off,2D cell 3) although it was found some cells with more persistent firing(seconds after light off, 2D cell 2). (similar to other studies, lagali,flannery, wt responses)

Sensitivity and Contrast Features of Restored LGN Responses

Current optogenetic strategies based on ChR (10¹⁵-10¹⁷ photons/cm²/s)and photosynthetic switches (10¹⁵-10¹⁸ photons/cm²/s) require high lightintensities for activation and long-term exposure of these lightintensities may be detrimental to the retina. In our experiments,restored neurons operate under a dynamic range of light intensitiesequivalent to natural daylight illumination (10¹³-10¹⁵ photons/cm²/s)(FIG. 6). Importantly robust, repeatable sustained and transient, ON andOFF responses were found across these light intensities, indicating howsensitive the system is. Some light responses were also found at lowerlight intensities (10¹² and 10¹¹, although these were not as readily/aswell/poorly reproducible (data not shown). No convincing light responseswere recorded at levels lower than 10¹⁵ photons/cm²/s when untreated rd1retinas were stimulated.

So far, studies on novel optogenetic tools have all characterisedrestored responses under dark-adapted conditions, using full field lightsteps from darkness. Although this increases the possibility of evokingstrong neuronal responses, these conditions are rarely present in reallife scenarios. The system was tested under more natural conditions, inlight adapted states. Robust, high amplitude, repeatable responses wererecorded under light adapted conditions, Michelson contrast 96% (FIG.6B). No contrast detecting cells were found in response to light adaptedstimulation of control rd1 retinas.

(Lower contrasts (66% and 33%,) also evoked measurable responses,although these were not as highly reproducible across repeated trials(see supplemental/or not show).

Restricting Ectopic Expression of Rod Opsin Using a Cell SpecificPromoter

The diversity of the generated visual code with unrestricted ectopicexpression of rod opsin was encouraging. However, it was decided toinvestigate if there would be any changes to this code if rod opsinexpression was restricted to “ON pathway” only. Therefore, rod opsin wasexpressed in ON bipolar cells using a cell specific Grm6 promoter (FIG.7A-C). FIGS. 8-10 show restored dLGN light responses under various lightconditions for mice that have been treated with rod opsin targeted toONBP cells. Sensitivity was demonstrated down to normal lightingconditions. Figures for irradiance quoted are for corneal measurements.Retinal irradiance will be approximately one order of magnitude lower.

In-vivo LGN recordings from these mice also show a significantlyincreased number of light responses (n=100) compared to internalcontrols (n=6) following stimulation with full field flashes (2 s) of410 nm light at 15.4 log photons/cm2/s. Once again, a range of responseprofiles was observed—sustained, transient, ON and OFF (excitatory andinhibitory) in the rod opsin treated group. Compared to untargetedresponses, light responses from targeted ON-BP cells were more evenlydistributed between ON and OFF types. Across the population of lightresponsive cells we observed 48/84 (57.1%) of ON responses and 36/84(42.9%) of OFF responses. These were broadly subdivided into ONsustained cells (34/84; 40.5%), ON transient cells (14/84; 16.6%), OFFexcitatory cells (6/84; 7.1%) and OFF inhibitory cells (30/84; 35.7%).More detailed characterisation of restored light responses showed fastON-transient, ON-sustained and OFF-transient responses (onset withinhundreds of ms) and slower OFF-inhibitory responses (onset withinseconds). These delayed robust OFF inhibitory responses were a specificfeature of targeted rod opsin expression (4G cell 2 and 3).

It was next investigated if the sensitivity and contrast detection ofON-bipolar driven responses is similar to our untargeted group.Comparable sensitivity profile of ON-bipolar driven responses was foundto the non-selective/untargeted group and light responses at 13.4 logphotons/cm2/s (FIG. 4H) were recorded. Similarly, in light adaptedconditions, we also found cells responsive to Michelson contrast level(96%, FIG. 41).

(Lower amplitude and less reproducible responses were recorded at lowercontrast levels (66% and 33%), similar to those observed from untargetedretinas (data not shown))

Light-Induced Behavioural Responses

Next, it was investigated whether the visual information transmitted tothe brain, driven by both untargeted and targeted ectopic rod opsintransduction, could restore lost visual functions in blind rd1 mice.First, a simple behaviour response to light, the pupillary light reflex(PLR) was tested. This reflex is normally mediated by melanopsinexpressing ipRGCs (Lucas R J, et al Science. 2003 Jan. 10;299(5604):245-7.) and is retained at high threshold in rd1 mice afterphotoreceptor degeneration (Lucas R J, et al Nat Neurosci. 2001 June;4(6):621-6., Lin B, Proc Natl Acad Sci USA. 2008 Oct. 14;105(41):16009-14.). Irradiance-response curves were recorded for maximumpupillary constriction during ten seconds of white light at a range oflight intensities (FIG. 11A). Impaired PLR was found in GFP injected,control mice, confirming previous findings. Unrestricted rod opsinexpression restored PLR comparable to wild type behaviour (FIG. 11A-D).However, with the targeted expression, the PLR remained largely impaired(FIG. 11A-C).

It was then asked whether the restored optogenetic visual code couldsupport more complex visual discrimination at behavioural level.Motivated by classical open-field light/dark box tests (Bourin M,Hascoët M (2003), Eur J Pharmacol 463(1-3): 55-65. the locomotorbehaviour in response to various artificial and natural light stimuliwas measured. Mice were placed in an open field modified light/dark boxand allowed free movement between two arenas via an opening in theseparating wall. Flat screen computer monitors outside each arena wereused to display a variety of visual stimuli. The light intensities ofour stimuli were equivalent of indoor lighting levels (range from0.00132 W/m2 for black screen to 0.116 W/m2 for white screen). Simpledark-light discrimination was first tested. After initial habituation tothe novel environment (3 min) mice were allowed to explore the box for 5minutes in dark (dark screen radiance=0.004072 Wsr⁻¹m⁻²;irradiance=0.00132 W/m2), followed by 5 minutes in white light (whitescreen radiance=0.06526 Wsr⁻¹m⁻²; irradiance=0.116 W/m2). The locomotoryassay was first evaluated in wild-type mice where activity plot shows asignificant increase in locomotion in the first 30 s of transition fromdark to light (FIG. 12, mean distance in dark=24.46±7.00 cm; meandistance in light=147.5±5.09 cm; p=0.0066). Blind rd1 mice showed nosignificant change in activity during this dark-light transition (meandistance in dark=91.75±7.70 cm; mean distance in light=100.2±8.145 cmp=0.5541). However, activity plots for the treated groups, rd1-hRho-CAGand rd1-hRho-grm6, display a sudden decrease in motor activity whenlight is turned on. This light-induced ‘freezing’ behaviour wassignificant in the first 30 s of transition from dark to light (forrd1-hRho-CAG, mean distance in dark=98.40±16.12 cm; mean distance inlight=69.73±13.32 cm; p=0.0070; for rd1-hRho-grm6 mean distance indark=112.9±21.47 cm; mean distance in light=68.37±16.81 cm; p=0.0224).

After establishing that the treated mice can distinguish light fromdark, their behaviour in response to more dynamic visual stimuli wastested. Locomotor activity in light adapted conditions (5 minutes ofexposure to gray screen light, radiance=0.03342 Wsr⁻¹m⁻²;irradiance=0.0663 W/m2) was compared to to full field flicker at 4 Hz(FIG. 13A). Rd1-hRho-grm6 group was able to resolve this flickerfrequency (mean distance in gray=151.3±34.95 cm; mean distance inflicker=83.7±13.76 cm; p=0.0466), whereas rd1-hRho-CAG and untreatedblind mice showed no change in behaviour (FIG. 13B). We then testedrd1-hRho-CAG at a lower frequency, at 2 Hz and found that they dorespond (likely limit of this system) (data not shown).

As the targeted rd1-hRho-grm6 mice had the best response with full fieldflicker this group was further tested to see if mice can discriminatemore subtle changes in light and detect contrast. Indeed it was showedthat mice responded at different Michelson contrasts (FIG. 14; meandistance in gray=179.3±17.34 cm; mean distance in flicker=127.2±16.29cm; p=0.0238 At 68.6% contrast and mean distance in gray=151.3±34.95 cm;mean distance in flicker=83.7±13.76 cm; p=0.0466 at 100% contrast).

Higher flicker frequencies in rd1-hRho-grm6 mice were also looked at, 10Hz and 20 Hz. No response at 20 Hz was found but convincing response at10 hz (FIG. 15; mean distance in gray=93.64±26.39 cm; mean distance inflicker=162.6±18.57 cm; p=0.039 at 10 Hz).

Responses to Naturalistic Scenes

Having described characteristics of restored responses using a varietyof artificial stimuli, it was set out to determine whether ectopic rodopsin could drive visual responses at brain level under morenaturalistic scenes with spatial properties. To this end, a naturalisticmovie was projected (mice moving around an open arena) to Rd1-CAG-hRhoand Rd1-grm6-hRho anaesthetised mice and recorded in-vivo responses fromthe LGN. The 30-second movie was presented repeatedly over a 30-minuteperiod allowing identification of cells with reproducible firingpatterns over many presentations. Individual neurons whose firing rateis modulated by particular features of the movie scene after stimulationof both untargeted and targeted retinas (FIG. 16A to C) were observed;no ‘movie-responsive’ neurons from the control eyes.

It was then investigated whether mice can see the naturalistic scenes?In an open field behavioural locomotory assay, a naturalistic moviefeaturing a looming owl was presented to the mice in the test box.Indeed it was found that the rd1-hRho-grm6 mice responded to a loomingowl (FIG. 6C) showing a significant change in locomotor behaviour in a30 s transition from uniform light to a movie scene (FIG. 14; meandistance in gray=67.68±22.05; mean distance in naturalmovie=110.2±19.44; p=0.0079). Interestingly they did not displayfreezing behaviour, but indeed increased their motor activity, likely inan attempt to escape from the predator. No changes were found inbehaviour in response to a movie in blind Rd1 mice or Rd1-hRho-CAG mice.

Materials and Methods

Animals

These mice are a model of severe and rapid form of retinal degeneration,similar to some forms of retinitis pigmentosa in humans (McLaughlin etal., 1993). All animal experiments were conducted in accordance with theUK Home Office regulations for the care and use of laboratory animals,the UK Animals (Scientific Procedures) Act and the Animal Welfare Bodyof the University of Manchester. Animals were kept under a 12 hourlight:dark cycle and supplied with food and water ad libitum.

Gene Delivery via AAV

Intraocular injections were carried out in mice at six weeks of ageunder general anaesthesia using isofluorane. Prior to injections, pupilswere dilated with tropicamide and phenylephrine. A custom madeultra-fine needle (Hamilton RN needle 34 gauge, supplied by ESSLAB) wasattached to a 5 μl Hamilton glass syringe and was passed at 45 degreesthrough the pars plana into the vitreous cavity, carefully avoiding thelens and blood vessels. The injection was performed under a directvisualisation of the needle tip through cover-slipped eyes under theoperating microscope. The vectors, rAAV serotype 2 (rAAV2/2, or simplyAAV2) expressing rod opsin or GFP under the control of a strongubiquitous pan-neuronal promoter (CAG) or ON-bipolar cell specific(Grm6) promoter were obtained from Vector Biolabs, Philadelphia, USA.The CAG promoter is a fusion of CMV early enhancer and chicken β-actinpromoter. The Grm6 promoter is a fusion of 200-base pair enhancersequence of the mouse Grm6 gene encoding for ON-bipolar cell specificmetabotropic glutamate receptor, mGluR6, and an SV40 eukaryoticpromoter. The gene of interest in each case was flanked by invertedterminal repeat (ITR) domains and stabilised by polyadenylation signalsequence (polyA) and a woodchuck hepatitis posttranscriptionalregulatory element (WPRE).

One eye of each mouse was injected with rod opsin expression construct(either AAV2-ITR-CAG-hRho-polyA-WPRE-ITR for untargeted expression orAAV2-ITR-grm6-hRho-polyA-WPRE-ITR for targeted expression) and the otherwith GFP expression construct (either AAV2-ITR-CAG-GFP-polyA-WPRE-ITRfor untargeted expression or AAV2-ITR-grm6-GFP-polyA-WPRE-ITR fortargeted expression). Each eye received 3 μl of viral constructcontaining 1×10¹³ genomic counts, in combination with 0.5 μl ofglycosidic enzyme solution containing 0.125 units of heparinise III(E.C. 4.2.2.8) and hyaluronan lyase (E.C. 4.2.2.1) obtained fromSigma-Aldrich, Dorset, UK. The enzyme solutions were made fresh on theday of injection by dissolving the enzymes in sterile phosphate-bufferedsaline (PBS). The vector and enzymes were mixed in a syringe immediatelybefore an eye injection and were given in a single combined injection.

Tissue Processing, Immunohistochemistry and Bioimaging

For tissue processing, retrieved eyecups (>6 weeks post vectorinjection) were fixed in 4% paraformaldehyde (PFA) for 24 hours at 4° C.The tissue was then washed in PBS and further fixed in 30% sucrose inPBS overnight at 4° C. Fixed eyes were cryo-protected in optimal-cuttingtemperature medium (Raymond A Lamb Ltd., Eastbourne, UK) and frozen at−80° C. until further processing. Cryo-protected retinal section werecut on a cryostat (Leica, Microsystems) horizontally through the eyecup@8-10 μm thickness from ventral to dorsal side, so that each sectioncontained a complete nasal to temporal cross-section of the retina.Ten-twelve sections were collected on each slide containing sectionsrepresentative of the entire retina. Slides were stored at −80° C.

For immunohistochemistry, slides were removed from freezer and allowedto air-dry at room temperature for 1 hour. Sections were permeabilisedby immersing slides in PBS with 0.2% Triton for 20 minutes at roomtemperature. Following this sections were background blocked with PBSwith 0.2% Triton X-100 containing 10% donkey serum (D9663; Sigma, UK)for 1 hour at room temperature. Primary antibody (Rabbit Anti-HumanRhodopsin, Abcam, Ab112576) was applied at 1:200 dilution in blockingbuffer (PBS with 0.2% Triton X-100 and 2.5% donkey serum) for 3 hours atroom temperature. After washing in tween 0.05% PBS, four times for 10minutes, sections were incubated with secondary antibody (Alexa Fluor®546 Donkey Anti-Rabbit IgG (H+L) Antibody, Life technologies, lot:1504518) diluted 1:200 in PBS with 0.2% Triton X-100 and 2.5% donkeyserum for 2 hours at room temperature. Slides were then washed fourtimes for 10 minutes in 0.05% tween PBS followed by one final wash withdH₂O. After removing excess fluid, slides were mounted with fluorescentmounting media containing DAPI (Vectashield, Vector Laboratories Ltd.,Peterborough, UK) to stain cell nuclei.

For bioimaging, sections were analyzed under an Olympus BX51 uprightmicroscope using ×4, ×10 and ×20 Plan Fln objectives and captured usinga Coolsnap ES camera (Photometrics, Tucson, Ariz.) through MetaVueSoftware (Molecular Devices Ltd. Wokingham, UK). Images were taken underspecific band pass filter sets and colour-combined images were used forfurther processing using ImageJ.

Multi-Electrode Array Recordings

Multi-electrode array recordings were performed on rod opsin treated rd1mice (n=6) and GFP injected rd1 controls (n=2). Enucleated eyes wereplaced in a petri dish filled with carboxygenated (95% CO2/5% CO2) aCSF(artificial cerebro-spinal fluid, concentration in mM: 118 NaCl, 25NaHCO₃, 1 NaH2PO4, 3 KCl, 1 MgCl2, 2 CaCl₂), 10 C6H1206, 0.5L-Glutamine). Retinas were then carefully isolated in diffuse red lightunder a dissecting microscope and mounted, ganglion cell down, onto a60-channel or 256-channel multi-electrode array (Multi Channel Systems,Reutlingen, Germany). Retinal explants were coupled in place with aweighted dialysis membrane, and continuously perfused withcarboxygenated aCSF at 2.2 ml per minute using a peristaltic pump(SCI400, Watson Marlow, UK), and maintained at 32° C. using a UniversalSerial Bus temperature controller regulating an inline heater for theinflow of aCSF. Light stimuli (white light) were presented by acustomised light engine source (Lumencor, USA). At brightest intensityLEDs were @15 Log photons/cm²/s. A National Instruments card (USB-6229)controlled by programmes written in LabVIEW (Version 8.6, NationalInstruments, TX, USA) was used to control stimulus duration andintensity by altering LED output and adjusting filter wheel containingneutral-density filters (Cairn Research Ltd). Stimuli were delivered at2-second pulses of light with 20 s inter-stimulus intervals for 20-30repeats at ND0 (15 Log photons/cm²/s) to ND4. Data were sampled at 25kHz frequency during the acquisition of both spontaneous and evokedactivity and recorded for off-line sorting using Offline Sorter(Plexon). After removing clear artefacts common to all channels,principal component analyses were used to discriminate single units,identified as distinct clusters of spikes within the principal componentspace, with a clear refractory period in the interspike intervaldistribution. Spike sorted, single unit data were then further analysedusing Neuroexplorer (Nex Technologies) and MATLAB R2010a (The MathworksInc.).

Pupillometry:

Pupillary light reflex (PLR) was measured in were performed on wild-typemice (6), rd1-CAG-GFP mice (n=16), rd1-CAG-hRho mice (n=10) andrd1-grm6-hRho (n=6) at six to eight weeks post injections. Mice weredark-adapted for 1 hour before the recordings. Light stimuli wereprovided by a quartz halogen lamp and were transmitted along afiber-optic bundle to an integrating reflective sphere, which provideduniform light at the mouse cornea. Consensual PLR was recorded inun-anaesthetised, lightly scruffed mice, under infra-red conditions withan infra-red sensitive CCD camera fitted with 10× macro lens and aninfra-red filter. An intervening shutter controlled stimulus timing. Asingle trial lasted 20 seconds: 5 seconds light OFF, 10 seconds lightON, 5 seconds light OFF. The intensity of the light was controlled byneutral density (ND) filters and mice were subjected to white lightexposures in an ascending intensity series, with individual trials beingseparated by at least 5 minutes. Photon emission values for NDs in logphotons/cm2/s ranged from 15.85 at ND0 to 10.85 at ND5. Pupillaryresponses were quantified from the video images, by using Virtual Duband ImageJ software and data were normalised to pupil area immediatelypreceding the light onset.

In-Vivo Electrophysiology:

In-vivo electrophysiology was performed on wild-type mice (n=2),rd1-CAG-hRho mice (n=7) and rd1-grm6-hRho (n=5). Six weeks postinjections and after measurement of PLR, mice were anaesthetised withurethane (intraperitoneal injection 1.7 g/kg; 30% w/v; Sigma Aldrich,Poole, UK). Animals were restrained in a stereotaxic frame (SR-15M;Narishige International Ltd, London, UK) and core body temperature wasmaintained at 37° C. via a homeothermic heat mat (Harvard Apparatus,Edenbridge, UK). Pupils were dialated with atropine and mineral oil(Sigma Aldrich) was applied to retain corneal moisture. A smallcraniotomy and durotomy (˜1 mm²) were performed directly above eachlateral geniculate nucleus (LGN) using stereotaxic coordinates accordingto mouse atlas (Paxinos and Franklin, 2001; hole centre=bregma: −2.46mm; midline: −2.8). A 32-channel electrode (NeuroNexus TechnologiesInc., MI, USA) was introduced to each LGN in the centre of the hole(medial shank: −2.5 mm relative to midline; depth: −2.6 mm relative tobrain surface at 18 degrees angle) for simultaneous recording from bothLGNs. Mice were left for 30 min prior to recordings, to dark adapt andto allow neuronal activity to stabilize following electrode insertion.Visual stimuli were provided by UV LED (Thorlab Amax: 405 nm) anddelivered via fiber optic to purpose-made eye cones tightly positionedonto each eye to minimise any potential light leak. A NationalInstruments card (USB-6229) controlled by programmes written in LabVIEW(Version 8.6, National Instruments, TX, USA) was used to controlstimulus duration and intensity by altering LED output and adjustingfilter wheel containing neutral-density (ND) filters (Cairn ResearchLtd). At brightest intensity (ND0) LEDs were@47 W/m2 or 15.4 logphotons/cm²/s of effective flux for rod opsin. Data were acquired usinga Recorder64 system (Plexon, Tex., USA) with signal amplification by a20× gain AC-coupled headstage (Plexon, Tex.) followed by preamplifierconditioning providing a total gain of 3500×. Data were high-pass (300Hz) filtered and time-stamped neural waveforms were digitizedsimultaneously from all channels at a rate of 40 kHz. Multiunit data wasthen stored for offline sorting and analysis as for the MEA datadescribed above. At brightest intensity LEDs were 15 Log photons/cm²/s.Stimuli were delivered according to a light protocol consisting of 2parts. Part 1 included flashes from darkness: 2 s light ON, 20 s lightOFF with 10 s offset between each eye. This paradigm was repeated 30× ateach ND filter ranging from dimmest (ND3=12.4 log photons/cm²/s) tobrightest (ND0=15.4 log photons/cm²/s). Longer stimulus length was alsoused for more sustained responses: 10 s ON, 30 s OFF with 15 s offsetbetween each eye. The paradigm was repeated 10-20× at ND3 to ND0.

Part 2 of the light protocol involved recording in light adaptedconditions where 5-second steps of light were applied to a steadybackground illumination at Michelson contrast of 96%. There was a20-second inter-stimulus interval and a 10-second offset between twoeyes. This paradigm was repeated ten times.

(Part 2 of the light protocol involved recording in light adaptedconditions where increasing light contrasts were applied to a steadybackground illumination. Michelson contrasts, 33%, 66% and 96% wereapplied in 5-second steps, with a 20-second inter-stimulus interval anda 10-second offset between two eyes. This paradigm cycle was repeatedten times).

Responses to a naturalistic movie were also recorded. For this, aseparate set of recordings (rd1-CAG-hRho mice; n=2 and rd1-grm6-hRho;n=3) was obtained from the dLGN contralateral to the rod opsin treatedeye. A different experimental set up was used, involving a digitalmirror device projector (DLP® LightCommander™; Logic PD Inc.), in orderto maximise the wavelegths used and intensity of the movie. Theintrinsic light engine of the projector was replaced with amultispectral LED light source containing four independently controlledLEDs (λ_(max) at 405 nm, 455 nm, 525 nm and 630 nm; Phlatlight PT-120Series (Luminus Devices)). Light from the LEDs was combined by a seriesof dichroic mirrors (Thorlabs), and directed onto the projector. Themovie was presented using Python running PsychoPy Version 1.70.00software. It featured mice moving around a behavioural arena includingmovement and looming of different sized objects (subtending visualangles ranging from 0.5° to 36°) at a range of orientations, speeds andcontrasts (maximum Michelson contrast=96%). The movie lacked differencesin colour, and changes in irradiance across time were minimal (standarddeviation of irradiance=5.94%). Previous validations in wild-type micehave shown undetectable responses for presentations of de-focussedversions, indicating that most activity was elicited by changes inspatial patterns and object motion.

Data were acquired using a Recorder64 system (Plexon, Tex., USA) withsignal amplification by a 20× gain AC-coupled headstage (Plexon, Tex.)followed by preamplifier conditioning providing a total gain of 3500×.Data were high-pass (300 Hz) filtered and time-stamped neural waveformswere digitized simultaneously from all channels at a rate of 40 kHz, andstored for offline analysis.

To confirm the location of recording sites, the recording electrode wasdipped in fluorescent dye (Cell Tracker CM-Dil; Invitrogen) prior toinsertion into the brain. After in-vivo recordings, the mouse's brainwas removed and post-fixed overnight in 4% paraformaldehyde, prior tocryoprotection for 24 hours in 30% sucrose. 100 μm coronal sections werethen cut using a sledge microtome, mounted onto glass slides and coverslipped using Vectashield (Vector Laboratories, Inc.).

Multi-Electrode Array (MEA) Recordings:

Immediately after in-vivo electrophysiology recordings mice wereeuthanised and enucleated. Eyes were placed in a petri dish filled withcarboxygenated (95% CO2/5% CO2) aCSF (artificial cerebro-spinal fluid,concentration in mM: 118 NaCl, 25 NaHCO₃, 1 NaH2PO4, 3 KCl, 1 MgCl2, 2CaCl₂), 10 C6H1206, 0.5 L-Glutamine). Retinas were then carefullyisolated in diffuse red light under a dissecting microscope and mounted,ganglion cell down, onto a 60-channel multi-electrode array (MultiChannel Systems, Reutlingen, Germany). Retinal explants were coupled inplace with a weighted dialysis membrane, and continuously perfused withcarboxygenated aCSF at 2.2 ml/minute using a peristaltic pump (SCI400,Watson Marlow, UK), and maintained at 32° C. using a Universal SerialBus temperature controller regulating an inline heater for the inflow ofaCSF. Light stimuli were provided by a customised light engine(Lumencor, USA) to present UV light (Amax: 405 nm). At brightestintensity LEDs were −15 Log photons/cm²/s. A National Instruments card(USB-6229) controlled by programmes written in LabVlEW (Version 8.6,National Instruments, TX, USA) was used to control stimulus duration andintensity by altering LED output and adjusting filter wheel containingneutral-density filters (Cairn Research Ltd). Stimuli were deliveredaccording to a light protocol as for in-vivo electrophysiology datadescribed above. Data were sampled at 25 kHz frequency during theacquisition of both spontaneous and evoked activity and recorded foroff-line analysis.

Behaviour

Currently, commonly used visual discrimination tasks require extensivetraining, stressful environments, or are based on reflexes rather thangoal oriented behaviour. To address these deficits, a simple, open-fieldbased method of assessing vision based on spontaneous behaviour has beendeveloped. Using a modification of the light/dark box (dimensions:length=40 cm width=40 cm and height=30 cm, open top) mice were allowedfree movement between two equal arenas (east and west halves) via anopening in the separating wall. Box was made of Perspex glass and itswalls were painted white except for the two long sides of each arena,which were kept clear. Two identical infra-red lamps were placedcentrally above each arena, to allow visualisation under darkconditions. The visual stimuli were displayed from two 17-inch flatscreen computer monitors, facing clear walls of each arena, using aDualHead2Go Digital Edition external multi-display adapter (MatroxGraphics Inc.). This allowed both the control and the test stimuli to beshown on separate monitors at the same time. A variety of visual stimuliwere displayed including a full field uniform light (black, gray andwhite), full field flicker and a naturalistic movie. These stimuli werecreated using a custom written programme that allowed the formation oftwo different stimuli in two different windows simultaneously, whilstretaining the ability to operate both individually. The programmeallowed the values of the white (255), black (0) and grey (128)components of the stimuli to be altered to achieve desired brightnessand contrast levels.

Behavioural experiments were performed on wild-type mice (5),rd1-CAG-GFP mice (n=6), rd1-CAG-hRho mice (n=6) and rd1-grm6-hRho (n=5).Before the experimental period, mice were handled and habituated totheir novel environment over 5 days, at the same time each day, in thefollowing manner: animals were brought into the testing room in theirhome cages, placed into the experimental box and allowed to move freelywith their littermates for 30 minutes.

Following the habituation period, behaviour experiments were conductedover several weeks in a completely dark room at the same time each day.Each group of mice was allowed to undergo only one testing condition perday. On each test day, mice were brought into the testing room in theirhome cages, allowed to accommodate to the testing room conditions for 30minutes and then each mouse was tested individually. Mice were placedinto the open field box (randomly to east or west half) and allowed tomove freely between two arenas. All test trials were recorded underinfra-red conditions through a camcorder fitted with an infra-red filter(λ=665 nm). The box was thoroughly cleaned with 70% ethanol after eachtest trial and allowed to air-dry before next mouse was placed into thebox.

A recording trial began after 3 minutes of habituation. Each trial runconsisted of 5 minutes of control stimulus, following which a teststimulus was presented on a screen facing an arena that contained amouse at this time point. For dark-adapted experiments, control stimulusconsisted of 5 minutes in dark (screens switched on dark;radiance=0.004072 Wsr⁻¹m⁻², irradiance=0.00132 W/m2) followed by 5minutes in light (white screen; radiance=0.06526 Wsr⁻¹m⁻²,irradiance=0.116 W/m2) and then 1 minute back in the dark.

For light adapted experiments, control stimulus consisted of 5 minutesin uniform gray screen light (gray screen; radiance=0.03342 Wsr⁻¹m⁻²,irradiance=0.0663 W/m2), followed by 5 minutes of full field flicker(flicker screen; radiance=0.03342 Wsr⁻¹m⁻², irradiance=0.0663 W/m2)followed by 1 minute in the uniform light. For contrast sensitivityexperiments, screen outputs were adjusted to allow for different testingcontrasts between 2 arenas (Michelson contrasts: 100%, 68.6%, 37.3%,13.7% and 0% contrast). For the naturalistic movie experiment, controlstimulus consisted of 5 minutes of uniform gray light followed by 1minute of movie presentation and 1 minute back to the control condition.The (colour) movie featured a looming owl.

The recorded trials were stored for off-line analysis using a videotracking software device (EthoVision® XT 10.1 Noldus, Tracksys Ltd.,UK). We analysed distance travelled by each mouse in the entire box andoutputted results in 30 second bins. The mouse's ability to see thevisual stimuli was assessed as a change in locomotor activity during a1-minute transition period from a control to the test stimulus (i.e. weanalysed the distance travelled in 30 s under the control condition justprior to the test stimulus, and the distance travelled in 30 s justafter the test stimulus). Two-way paired t test was used for comparisonswithin the same group of mice before and after a test stimuluspresentation during a 1-minute transition period.

In summary the introduction of human rhodopsin into the inner retinalcells of mice that were blind from a severe retinal degeneration andloss of rod and cone photoreceptors resulted in the restoration ofvision as evidenced by a restoration in pupil responses and visualresponses in the brain (lateral geniculate nucleus) and retina.

Discussion

In this study it has been demonstrated that the expression of human rodopsin can bestow light sensitivity to blind retinas through a diversevisual code involving ON, OFF, transient and sustained pathways. Robustvisual responses were observed in vitro and in vivo under lightintensities equivalent of endogenous cone/PR vision and were able todrive sophisticated retinal circuit functions such as contrastdetection. Treated blind mice showed restored light-induced locomotoractivity under illuminance typical of natural indoor environments andwere able to resolve low frequency flicker in light adapted conditions.In addition, with targeted rod opsin treatment mice detected lowercontrasts, were able to resolve higher frequency full field flickers anddetect naturalistic movie scenes.

Introduction of microbial opsin channels and pumps and chemicalphotoswitches to surviving retinal neurons can restore lightsensitivity. Here, gene therapy can be used to deliver human rod opsinto blind mice. However, one of the main challenges in optogenetictherapy is to achieve stable and effective level of opsin expression inthe retina through a clinically relevant delivery method. Current viraldelivery methods require invasive sub-retinal injections wheretransduction is limited to a small portion of the retina at the site ofinjection. More global transduction could be achieved by injection intothe vitreous, a much safer technique that is commonly used in routineophthalmic practice. However, physical barriers created by theextracellular matrix of retinal cells limit viral transduction from thisroute. Several novel mutant AAV vectors have emerged (4YF and 7m8) withability to penetrate deeper into retinal tissue but currently requirevery high viral titers (10¹⁴) with potentially high immunogenicity andoff target effects. In the present approach intravitreal delivery ofAAV2 vector has been used, which has already been proven safe inclinical trials. In addition, a combination of enzymes targetingextracellular matrix proteins that disrupt the physical barriers toviral particles reaching the retina from the vitreous has beenidentified. Co-injecting these enzymes greatly increases the number andspatial spread of neurones infected by the virus.

In the intact retina mammalian rod opsin is normally found inspecialised rod photoreceptors. It belongs to a family of a G-proteincoupled receptors (GPCRs) that functions through signalling cascadeintermediates. On photoactivation, rod opsin couples to transducin (Gt;a member of Gi/o subfamily) for visual signal transduction (Palczewski,K. G. Annu. Rev. Biochem. (2006) 75, 743-767 in vivo, leading toincrease in K+ currents, membrane hyperpolarization and inhibition ofneurotransmitter release. A sign inverting synapse betweenphotoreceptors and their second order neurons, bipolar cells, convertsthis inhibitory response into positive electrical signal that istransmitted via the ganglion cells to the brain for visual percepts.

Opsin channels (ChR) and pumps (HaloR) have been functionally expressedin blind retina leading to depolarization and hyperpolarizationrespectively. In addition, vertebrate rod opsin has been expressed incell culture and in vivo outside the retina (cerebellar purkinje cells)and was shown to inhibit neuronal excitability when activated by light(Li X, et al Proc Natl Acad Sci USA. 2005 Dec. 6; 102(49):17816-21;Gutierrez D V, et al J Biol Chem. 2011 Jul. 22; 286(29):25848-58.).However it was now known that a GPCR like rod opsin would be able tofunction outside native photoreceptors when expressed directly in secondor third order neurons (RGCs) and exert its inhibitory action to producevisual signal. It was reasoned that in blind retinas, decoupled fromnormal photoreceptor input, there is an overall increase in basalactivity of surviving output neurons and that rod opsin could act tosuppress this ‘hyperactivation’ and improve the signal to noise ratio inorder to support visual discrimination.

Here, it is shown that ectopic rod opsin can express in the innerretinal cells and function outside native photoreceptors. Indeed aspredicted it was observed that inhibitory OFF responses in vivo (firingrate decreases with light ON stimulation) in both targeted (35.7% of alllight responses) and untargeted (10.8%) rod opsin treatments. These arenot normally seen with WT responses and we also did not observe suchresponses in our in-vitro preparations. However, suprisingly, manypositive ‘ON responses’ were found both in-vitro and in-vivo. Indeed adiverse set of responses were found with both untargeted and targetedtreatments including ON sustained, ON transient, and OFF excitatory(firing rate increases with light OFF) responses, as well as in-vivo OFFinhibitory signals. In addition, a small number of ON-OFF responsesin-vivo were observed with untargeted treatment. This would suggest thatrod opsin can modulate cell behaviour in both depolarising andhyperpolarising light dependent fashion. This would suggest that rodopsin expression in ON bipolar cells is strong enough to drivepost-synaptic third order neurons and stimulate amacrine inhibitoryloops (All cells) leading to activation of OFF bipolar cells andtherefore double inhibition or excitatory ON responses.

In intact systems visual information is processed through two parallelpathways, ON pathway and OFF pathway. In ON pathway, cells respond tolight increments, whereas in OFF pathway cells respond to lightdecrements. Our restoration of ON and OFF pathways in blind retinas isin support of other studies that have previously specifically targetedChR and LiGluR to ON-BP cells. However, previous studies usingubiquitous promoters (Lin B, et al Proc Natl Acad Sci USA. 2008 Oct. 14;105(41):16009-14) or specifically targeting RGC only, have reportedelectrophysiologically more uniform signals (e.g. sustained ON withectopic melanopsin) and no simultaneous restoration of both ON and OFFpathways (ChR in RGC drove only ON pathways, whereas HaloR in RGC onlyOFF pahways). It is possible that expression in RGCs only was not strongenough to directly activate the normal excitatory and inhibitory inputsto the cells. In addition in this case, even with the untargetedtreatment, it was found that rod opsin expression in INL cells (likelyBP cells), which could explain secondary activation of All loop anddissection of responses into both inhibitory and excitatory signals.

One of the main advantages of using rod opsin as an optogenetic tool torestore vision lies in its simplicity to provide self-containedphotoreception with its ability to use endogenous chromophore (retinalor cis-retinaldehyde) as the natural photoswitch. However, for visualresponses, rod opsin requires constant recycling of retinal, which isnormally bleached by light. This recycling is thought be dependent uponthe intimate contact between the rods and the RPE. Indeed, consistentwith previous studies (Li X, et al Proc Natl Acad Sci USA. 2005 Dec. 6;102(49):17816-21; Gutierrez D V, et al J Biol Chem. 2011 Jul. 22;286(29):25848-58), it was found that in vitro rod opsin bleaches readilyafter light stimulation and it requires constant exogenous supply ofchromophore (9-cis retinal) in order to sustain light dependentresponses. However, in vivo did not found bleaching to be a significantproblem. Importantly, many light responses were robust and repeatableover multiple trials. It is therefore likely that the degeneratingretina provides a good endogenous supply of retinal, and in the absenceof photoreceptors that normally take it up, rod opsin in inner retinalcells has access to the chromophore recycling by the RPE or Muller cellsto produce visual responses.

Another important aspect of rod opsin therapy is its ability to functionunder physiological light conditions, unlike current strategies based onmicrobial opsins and synthetic photoswitches that require extremely highlight intensities for activation. It was found that that ON and OFF,transient and sustained cell types function in vivo under a dynamicrange of light intensities tested (10¹³-10¹⁵ photons/cm²/s) fallingunder a range of endogenous cone sensitivity. Previously reportedstudies used much higher light intensities for activation: LiGluR>1.7×10¹⁷ photons/cm²/s (Gaub B M, et al 2014, Proc Natl Acad Sci USA.2014 Dec. 23; 111(51):E5574-83), ChR >3×10¹⁵-10¹⁷ photons/cm²/s (LagaliP S, et al 2008, Nat Neurosci. 2008 June; 11(6):667-75;) HaloR in RGCs>5.1×10¹⁸ photons/cm²/s (Zhang Y, et al J Neurosci. 2009 Jul. 22;29(29):9186-96) and HaloR in cone inner segments >10¹⁶ photons/cm²/s(Busskamp V, et al 2010, Science. 2010 Jul. 23; 329(5990):413-7.).Sensitivity is a factor of intacellular mechanisms and our data suggeststhat rod opsin can function through GPCR light amplification cascadeunder low light intensities. This is in contrast to microbial ionchannels (ChR2), pumps (HaloR) or photoswitches (LiGluR), which areunable to amplify signals at the protein level.

Further characterisation of the kinetics of restored responses in-vivoshowed that the onset, offset and the duration of the light responsevaried among cell types for both untargeted and targeted approaches. Itwas found that responses of varied latencies; some fast some slowerconsistent with previous studies (Caporale N, et al 2011, Mol Ther. 2011July; 19(7):1212-9; Gaub B M, et al 2014, Proc Natl Acad Sci USA. 2014Dec. 23; 111(51):E5574-83). Rod opsin light switch is intrinsically fastand slower light responses might be an effect of the dynamics of theintracellular signalling cascade and dependent upon native cell typeexpressing rod opsin. In addition, slower response onset is likely to bedue to non-saturating light input and lower level of expression of rodopsin in some cells. (Some fast rod opsin responses may be through fastkinetics of native mgluR6 receptor cascade in ONBP cells.)

The eye is sensitive over a wide range of light intensities fromscotopic (night) to photopic (day) vision. This sensitivity can bemeasured by minimum threshold intensity necessary to evoke vision. Oneof the important aspects of our vision is how quickly the eye recoversits sensitivity in dark after bright light exposures (dark adaptation)or more importantly how quickly it adapts to background illumination tobe able to discriminate objects in this background (light adaptation).So far most optogenetic studies have studied simple visual stimuliinvolving light steps from darkness. This is rather an un-naturalscenario and in the real world objects have contrast, which is constantand independent of ambient luminance. It was therefore questionedwhether the ectopic rod opsin could work under light adapted conditionsand conserve the restored visual code against changes in irradiance.Indeed robust dLGN responses were found after increment test lightstimulus presentations against a background illumination. (It ispossible that the observed robust segregation into ON and OFF pathwaysis able to facilitate this light adaptation and enhance contrastsensitivity).

Both untargeted and targeted expression was investigated. First anon-selective, untargeted approach with strong pan-neuronal promoter,CAG was used to deliver genes across the surviving inner retina. It wasnot known which neurons, if any, would express rod opsin and if it wouldexpress in one particular type of neuron more than the other or havepreference for either ON or OFF pathway or target both equally. It washoped that the imbalance in expression between ON and OFF pathways wouldmean that even though some signals would cancel each other out, therewould be an overall increase in visual information transmitted to thebrain. Advantage of this approach would also mean that the retinalganglion cells, which survive the longest (Mazzoni F,) J Neurosci28(52):14282-14292), are also targeted and this approach can be used inlate degeneration where bipolar cells become compromised (cronin).Second, it was tested whether restricting the expression to just ‘ON’pathway would lead to an improvement in visual signal bothelectrophysiologically and behaviourally. Rod opsin was specificallytargeted to retinal ON bipolar cells using a minimal cell specificpromoter Grm6 (Masu M et al., Cell 1995). Striking electrophysiologicalsimilarity was observed between untargeted and targeted rod opsintreatments. The recovered code was equally complex and diverse. Responsekinetics, the sensitivity and the contrast response were similar for thetwo treatments. Interestingly, targeted treatment led to more OFFresponses (mainly OFF inhibitory). However, there were importantdifferences behaviourally. First, differences in restoration of simplevisual function, PLR, was observed. Non-selectively expressed rod opsinrestored the drive to the RGC pathway mediating the PLR to near WTlevel. This is consistent with melanopsin and first generation LiGluR inRGC, which lead to near complete (Lin B, Proc Natl Acad Sci USA. 2008Oct. 14; 105(41):16009-14.) or partial (Caporale N, et al 2011, MolTher. 2011 July; 19(7):1212-9;) restoration of PLR.. Interestingly, whenexpression was restricted to ON-BP cells this pathway remained largelyimpaired. It is possible that untargeted expression of rod opsindirectly activated ipRGCs (coupling to the Gs/Gq cascade) leading tostimulation, whereas selective expression in ON-BP cells bypassed thedirect activation of the pathway and recovery of this non-visual reflex.

What about image forming pathways? Can this visual code that we createdbe used to improve image-forming vision under physiological lightlevels? One of the advantages of targeting specifically more distalretinal neurons would be to preserve inner retinal processing, fine-tunerestored responses into more coherent signals and achieve better qualityvision. So far optogenetic studies have addressed simple visual tasksinvolving dark-light discrimination and none have reported if the systemcould track changes against a background illumination or in response tonaturalistic scenes. It was found that restored in-vivo responses couldsupport simple dark-light discrimination with both treatments underillumination equivalent of indoor lighting. In addition, mice treatedwith untargeted and targeted rod opsin treatment could resolve fullfiled light flicker at 2 Hz against uniform background illumination. Inaddition it was found that mice with targeted treatment resolved higherfrequency flickers including 4 Hz and 10 Hz against uniform backgroundillumination. Moreover, mice with specific expression of rod opsin inONBP cells were able to detect 4 Hz flicker at a lower contrast levelagainst background illumination (Michelson contrast 66.7%).

Humans excel in processing of natural visual scenes. Despite constantlychanging visual scenes our brain is able to transforms complex patternsof light falling on our retina, and extract relevant information into acoherent percept within a few hundred milliseconds. With translationalpotential in mind, it was questioned whether ectopic rod opsin coulddrive signals robust enough to preserve many levels of visual processingcreating a visual code that mice could use to track naturalspatio-temporal modulations in light intensity at levels typical ofindoor illumination. Electrophysiologically, with both treatments, itwas set out of identify individual neurons that can track changes inlight levels over space and time that occur in natural movie scenes.This would suggest that the visual system (with its plasticity) is ableto exploit temporally and spatially pooled restored responses carryingsimple information on ON and OFF signals and contrast in order toprocess real natural scenes. In addition, we found that mice posttargeted rod opsin treatment increased their locomotory behaviour inresponse to natural movie scenes. This would suggest that the quality ofperceptual vision is improved with targeted compared to untargetedtreatment perhaps due to more coherent signal convergence from bipolarcells to RGCs necessary for more complex temporo-spatial discrimination.

In summary, the results show that human rod opsin in surviving innerretinal neurons is a promising strategy to restore vision in retinaldegeneration due to loss of photoreceptors and reverse advanced stagesof blindness. As a human protein it offers advantages over currentmicrobial-based therapies in terms of ethical and safety concerns. Itprovides a self-contained photoreceptive strategy that amplifies signalthrough intracellular cascade and is able to impart improved sensitivitycompared to current optogenetic strategies. When ectopically expressedin inner retinal neurons, it generates a diverse visual code based onboth ON and OFF responses capable of tracking changes in lightintensities over background illumination and in natural movie scenes.Behaviourally, treated blind mice are able to discriminate dark andlight and can resolve 2 Hz full field flicker in light adaptedconditions under illuminance typical of indoor room lighting. Inaddition, restricting the expression to ON BP cells, led to improvementsin visual percepts in vivo and treated mice were able to resolve higherfrequency flickers, detect lower contrast and resolve natural scenes.

1-81. (canceled) 82-90. (canceled)
 91. A composition comprising anucleic acid vector comprising a nucleic acid sequence encoding a humanrhodopsin or a human photopsin, wherein the nucleic acid is undercontrol of a promoter which directs expression of said nucleic acid toan inner retinal cell in a subject with a condition.
 92. A compositionaccording to claim 91, wherein the composition is an injectable liquid.93. The composition of claim 91, further comprising an extracellularmatrix degradation enzyme or an active fragment thereof.
 94. Thecomposition of claim 93, comprising a first injectable liquid comprisingthe nucleic acid sequence encoding human rhodopsin or human photopsin;and ii) a second injectable liquid comprising the extracellular matrixdegradation enzyme.
 95. The composition of claim 94, wherein the firstinjectable liquid is administered to the subject prior to anadministration of the second injectable liquid.
 96. The composition ofclaim 94, wherein the first injectable liquid is administered to thesubject subsequent to an administration of the second injectable liquid.97. The composition of claim 94, wherein the first injectable liquid isadministered to the subject simultaneously with an administration of thesecond injectable liquid.
 98. The composition of claim 93 wherein theextracellular matrix degradation enzyme is capable of degrading aglycosaminoglycan.
 99. The composition of claim 93, wherein theextracellular matrix degradation enzyme or an active fragment thereof isselected from the group consisting of a collagenase, hyaluronan lyase,heparinase I, heparinase II, heparinase III, chondroitin ABC lyase,chondroitin AC lyase, a metalloproteinase, an ADAMTS, a plasmin (serineprotease plasmin or its truncated form microplasmin (Ocriplasmin)),neutrophil elastase and cathepsin G, neuraminidase, N-glycanase,O-glycanase, and pronase.
 100. The composition of claim 93, wherein theextracellular matrix degradation enzyme or an active fragment thereofcomprises a combination of two or more extracellular matrix degradationenzymes.
 101. The composition of claim 100, wherein the extracellularmatrix degradation enzyme or an active fragment thereof comprises acombination of hyaluronan lyase and heparinase III.
 102. The compositionof claim 93 wherein an active fragment shares at least 70% sequenceidentity with a native extracellular matrix degradation enzyme.
 103. Thecomposition of claim 91, wherein the vector is a viral vector.
 104. Thecomposition of claim 103, wherein the viral vector is an AAV vector.105. The composition of claim 104, wherein the AAV vector comprises anAAV serotype 2 vector.
 106. The composition of claim 105, wherein theAAV vector comprises an AAV 4YF vector.
 107. The composition of claim105, wherein the AAV serotype 2 vector comprises an AAV 7m8 vector. 108.The composition of claim 91, wherein the promoter is selected from thegroup consisting of L7, thy-1, recoverin, calbindin, human CMV, GAD-67,chicken beta-actin, Grm6, Grm6 enhancer-SV40 fusion protein.
 109. Thecomposition of claim 108, wherein the promoter comprises a Grm6enhancer-SV40 fusion protein.
 110. The composition of claim 91, whereinthe inner retinal cell is a rod cell, a cone cell, an ON-bipolar cell,an OFF-bipolar cell, a horizontal cell, a ganglion cell, or an amacrinecell.
 111. The composition of claim 91, wherein the condition is aretinal dystrophy including a rod dystrophy, a rod-cone dystrophy, acone-rod dystrophy, a cone dystrophy and a macular dystrophy; anotherform of retinal or macular degeneration, an ischemic condition, uveitis,edema, or any other disease resulting from loss of photoreceptorability.
 112. The composition of claim 91, wherein the human photopsinis selected from the group consisting of Long Wavelength Sensitive(OPN1LW) Opsin, Middle Wavelength Sensitive (OPN1MW), and ShortWavelength Sensitive (OPN1SW) Opsin.
 113. The composition according toclaim 91, wherein the nucleic acid sequence comprises I) the rhodopsin(RHO) gene, or a fragment or derivative thereof, or ii) the Cone Homosapiens opsin 1, long wave sensitive OPN1LW gene, or a fragment orderivative thereof; or iii) the Cone Homo sapiens opsin 1: medium-wavesensitive OPN1MW, or a fragment or deriva-tive thereof; or iv) the ConeHomo sapiens opsin 1, short-wave-sensitive (OPN1 SW), or a fragment orderivative thereof.
 114. The composition according to claim 91 whereinthe nucleic acid sequence encoding human rhodopsin comprises thesequence of Genbank Accession No. BC111451.3 or a fragment or derivativethereof, or a sequence having at least 70% sequence identity thereto.115. A vector according to claim 91 wherein the vector further comprisesone or more regulatory sequences which direct expression to an innerretinal cell.
 116. A composition according to claim 93 wherein the firstand second injectable liquids are provided in separate containers.